Adverse side-effects associated with administration of an anti-hyaluronan agent and methods for ameliorating or preventing the side-effects

ABSTRACT

Provided herein are methods for ameliorating an adverse effect of systemic administration of a PEG hyaluronan degrading enzyme to a subject. The methods involve systemically administering a PEGylated hyaluronan degrading enzyme, particularly a PEGylated hyaluronidase, such as any of the animal or bacterial hyaluronidases, to the subject and administering an amount of a corticosteroid sufficient to ameliorate the adverse effect. Also provided are method of treating a hyaluronan-associated disease or condition for single-agent therapy or combination therapy.

RELATED APPLICATIONS

Benefit of priority is claimed to U.S. Provisional Application Ser. No.61/399,993, entitled “ADVERSE SIDE-EFFECT ASSOCIATED WITH ADMINISTRATIONOF PEGYLATED HYALURONIDASE AND METHODS FOR AMELIORATING OR PREVENTINGTHE SIDE-EFFECTS,” filed on Jul. 20, 2010 to Harold Michael Shepard,Curtis Thompson, Ziaoming Li and Gregory I. Frost, and to U.S.Provisional Application Ser. No. 61/455,260, entitled “ADVERSESIDE-EFFECTS ASSOCIATED WITH ADMINISTRATION OF ANTI-HYALURONAN AGENTSAND METHODS FOR AMELIORATING OR PREVENTING THE SIDE-EFFECTS,” filed onOct. 14, 2010, to Harold Michael Shepard, Curtis Thompson, Ziaoming Liand Gregory I. Frost.

This application is related to International Application No.PCT/US2011/044281, filed the same day herewith, entitled “ADVERSESIDE-EFFECTS ASSOCIATED WITH ADMINISTRATION OF ANTI-HYALURONAN AGENTSAND METHODS FOR AMELIORATING OR PREVENTING THE SIDE-EFFECTS,” whichclaims priority to U.S. Provisional Application Ser. Nos. 61/399,993 and61/455,260. The subject matter of the above-noted related application isincorporated by reference in its entirety.

This application is related to U.S. application Ser. No. 12/386,222,filed Apr. 14, 2009, which is published as US2010003238, which claimspriority to U.S. Provisional Appl. Nos. 61/124,278, 61/130,357 and61/195,624. This application also is related to International PCTApplication No. PCT/US2009/002352, filed Apr. 14, 2009, which ispublished as WO2009128917, and which also claims priority to U.S.Provisional Appl. Nos. 61/124,278, 61/130,357 and 61/195,624.

The subject matter of each of the above-referenced applications isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on Jul. 15, 2011, is identical, 824 kilobytes in size, andtitled P3084seq.001.txt.

FIELD OF THE INVENTION

Provided are methods associated with the use of anti-hyaluronan agents,such as hyaluronan degrading enzymes, for single-agent therapy orcombination therapy.

BACKGROUND

Hyaluronan (hyaluronic acid; HA) is a polysaccharide that is found inthe extracellular matrix of many tissues, especially in soft connectivetissues. HA also is found predominantly in skin, cartilage, and insynovial fluid in mammals. Hyaluronan also is the main constituent ofthe vitreous of the eye. HA has a role in various physiologicalprocesses, such as in water and plasma protein homeostasis (Laurent T Cet al. (1992) FASEB J 6: 2397-2404). Certain diseases and disorders areassociated with expression and/or production of hyaluronan.Hyaluronidases are enzymes that degrade hyaluronan. By catalyzing thebreakdown of HA, hyaluronidases can be used alone or in combination withother therapeutic agents to treat diseases or disorders associated withaccumulation of HA or other glycosaminoglycans.

SUMMARY

Provided herein are methods for ameliorating or preventing an adverseeffect in a subject from an administered anti-hyaluronan agent byadministering an amount of a corticosteroid to the subject sufficient toameliorate the adverse effect. The corticosteroid can be aglucocorticoid. For example, the glucocorticoid can be a cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone orprednisone, and in particular is a dexamethasone. In one example, thecorticosteroid is administered orally.

In some examples of the methods provided herein, the adverse effect is amusculoskeletal side effect. For example, the adverse effect is muscleand joint pain, stiffness of upper extremities, stiffness of lowerextremities, cramping, myositis, muscle soreness and tenderness over theentire body, weakness, fatigue and a decrease in range of motion at kneeor elbow joints. Typically, the adverse effect is one that results in adose-limiting toxicity (DLT) of the anti-hyaluronan agent. In themethods, the corticosteroid is administered in an amount to eliminate orincrease dose that results in the DLT of the anti-hyaluronan agent. Inanother example, the adverse effect is a Grade 3 on a toxicity scale andthe corticosteroid is administered in an amount to reduce the Grade toGrade 1 or Grade 2 or to Grade 3 that resolves within hours ofadministration of the corticosteroid.

In the methods, the anti-hyaluronan agent that is administered isadministered orally, intravenously (IV), subcutaneously,intramuscularly, intra-tumorally, intradermally, topically,transdermally, rectally or sub-epidermally. Generally, theanti-hyaluronan agent is a hyaluronan degrading enzyme or is an agentthat inhibits hyaluronan synthesis. For example, the anti-hyaluronanagent is an agent that inhibits hyaluronan synthesis such as a sense orantisense nucleic acid molecule against an HA synthase or is a smallmolecule drug. For example, an anti-hyaluronan agent is4-methylumbelliferone (MU) or a derivative thereof, or leflunomide or aderivative thereof. Such derivatives include, for example, a derivativeof 4-methylumbelliferone (MU) that is 6,7-dihydroxy-4-methyl coumarin or5,7-dihydroxy-4-methyl coumarin. In other examples, the anti-hyaluronanagent is a hyaluronan degrading enzyme that is modified by conjugationto a polymer. The polymer can be a PEG and the anti-hyaluronan agent aPEGylated hyaluronan degrading enzyme.

Provided herein are methods for ameliorating an adverse effect ofsystemic administration of a PEG hyaluronan degrading enzyme to asubject. The methods involve systemically administering a PEGylatedhyaluronan degrading enzyme, particularly a PEGylated hyaluronidase,such as any of the animal or bacterial hyaluronidases, to the subjectand administering an amount of a corticosteroid sufficient to amelioratethe adverse effect. In one example of the provided methods, thecorticosteroid is administered orally. In another example, PEGylatedhyaluronan degrading enzyme is administered intravenously.Administration of the corticosteroid and/or the PEGylated hyaluronandegrading enzyme can be effected systemically by any suitable route,such as, for example, intravenously, orally or intramuscularly.

In the provided methods, the adverse effects associated withadministration of a PEGylated hyaluronan degrading enzyme aremusculoskeletal side effects. Musculoskeletal side effects include, forexample, muscle and joint pain, stiffness of upper and lowerextremities, cramping, myositis, muscle soreness and tenderness over theentire body, weakness, fatigue and/or a decrease in range of motion atknee and elbow joints.

In some examples, the corticosteroid is a glucocorticoid. For example,the corticosteroid is a glucocorticoid selected from among cortisones,dexamethasones, hydrocortisones, methylprednisolones, prednisolones andprednisones. In a particular example, the glucocorticoid is adexamethasone.

In the provided methods, the corticosteroid is administered prior to,concurrent with, intermittently with or subsequent to administration ofthe PEGylated hyaluronan degrading enzyme. In one embodiment of themethods, the corticosteroid is co-administered with the PEGylatedhyaluronan degrading enzyme. For example, the corticosteroid isadministered prior to administration of the PEGylated hyaluronandegrading enzyme. In this example, the corticosteroid is administered ator about 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours or more prior tothe administration of the PEGylated hyaluronan degrading enzyme. In aparticular example, administration of the corticosteroid is at least ator about 1 or more hours prior to administration of the PEGylatedhyaluronan degrading enzyme.

In another embodiment of the methods, the corticosteroid is administeredsubsequent to administration of the PEGylated hyaluronan degradingenzyme. For example, the corticosteroid is administered at or about 0.5minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 18 hours, 24 hours or more after the administration of thePEGylated hyaluronan degrading enzyme. In another example,administration of the corticosteroid is at least 8 hours to 12 hoursafter administration of the PEGylated hyaluronan degrading enzyme.

In yet another embodiment of the methods, the corticosteroid isadministered prior to and after administration of the PEGylatedhyaluronan degrading enzyme. For example, the corticosteroid isadministered one to five minutes immediately before administration ofthe PEGylated hyaluronan degrading enzyme and eight hours afteradministration of the PEGylated hyaluronan degrading enzyme. In anotherexample, the corticosteroid is administered one hour beforeadministration of the PEGylated hyaluronan degrading enzyme and eight totwelve hours after administration of the PEGylated hyaluronan degradingenzyme. In the methods provided herein, any dosing regime iscontemplated as long as the time of dosing of the corticosteroidameliorates the one or more side effects associated with administrationof the PEGylated hyaluronan degrading enzyme.

In some embodiments of the methods, the corticosteroid is administeredin an amount between at or about 0.1 to 20 mgs, 0.1 to 15 mgs, 0.1 to 10mgs, 0.1 to 5 mgs, 0.2 to 20 mgs, 0.2 to 15 mgs, 0.2 to 10 mgs, 0.2 to 5mgs, 0.4 to 20 mgs, 0.4 to 15 mgs, 0.4 to 10 mgs, 0.4 to 5 mgs, 0.4 to 4mgs, 1 to 20 mgs, 1 to 15 mgs or 1 to 10 mgs. In a particularembodiment, the corticosteroid is administered in an amount between ator about 0.4 to 20 mgs. Exemplary doses of corticosteroid are any dosethat ameliorates the one or more side effects associated withadministration of the PEGylated hyaluronan degrading enzyme.

In one embodiment of the methods, the PEGylated hyaluronan degradingenzyme is administered in an amount of at or about 0.0005 mg/kg, 0.0006mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.0016mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg,0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.016 mg/kg, 0.02mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.30mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg,1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5mg/kg, 9 mg/kg, 9.5 mg/kg or 10 mg/kg of the mass of the subject to whomit is administered.

In another embodiment of the methods, the PEGylated hyaluronan degradingenzyme is administered in an amount of at or about 10 Units/kg (U/kg),16 U/kg, 32 U/kg, 64 U/kg, 100 U/kg, 200 U/kg, 300 U/kg, 400 U/kg, 500U/kg, 600 U/kg, 700 U/kg, 800 U/kg, 900 U/kg, 1,000 U/kg, 2,000 U/kg,3,000 U/kg, 4,000 U/kg, 5,000 U/kg, 6,000 U/kg, 7,000 U/kg, 8,000 U/kg,9,000 U/kg, 10,000 U/kg, 12,800 U/kg, 20,000 U/kg, 32,000 U/kg, 40,000U/kg, 50,000 U/kg, 60,000 U/kg, 70,000 U/kg, 80,000 U/kg, 90,000 U/kg,100,000 U/kg, 120,000 U/kg, 140,000 U/kg, 160,000 U/kg, 180,000 U/kg,200,000 U/kg, 220,000 U/kg, 240,000 U/kg, 260,000 U/kg, 280,000 U/kg,300,000 U/kg, 320,000 U/kg, 350,000 U/kg, 400,000 U/kg, 450,000 U/kg,500,000 U/kg, 550,000 U/kg, 600,000 U/kg, 650,000 U/kg, 700,000 U/kg,750,000 U/kg, 800,000 U/kg of the mass of the subject to whom it isadministered.

In a particular embodiment of the method, the PEGylated hyaluronandegrading enzyme is administered in an amount between at or about 10 and320,000 Units/kg of the mass of the subject and the corticosteroid isadministered in an amount between at or about 0.4 to 20 mgs.

In some embodiments of the method, the PEGylated hyaluronan degradingenzyme used in the methods is a hyaluronidase, for example, any animalor bacterial hyaluronidase. In some examples, the hyaluronidase is asoluble hyaluronidase. For example, the hyaluronidase is soluble PH20hyaluronidase that is selected from among a human, monkey, bovine,ovine, rat, mouse or guinea pig PH20. In a particular example, thehyaluronidase is human PH20. In other examples, the hyaluronidase is ananimal-derived hyaluronidase. For example, hyaluronidase is ananimal-derived hyaluronidase selected from among a purified bovinetesticular hyaluronidase or a purified ovine testicular hyaluronidase.In a particular aspect of the method, the PEGylated hyaluronan degradingenzyme is PEGylated PH20 (PEGPH20) and the corticosteroid isdexamethasone.

In one embodiment, the hyaluronidase used in the methods provided hereinis neutral active and N-glycosylated. In some aspects, the neutralactive and N-glycosylated hyaluronidase polypeptide is a full-lengthPH20 or is a C-terminal truncated form of the PH20, wherein thefull-length PH20 has the sequence of amino acids set forth in SEQ IDNO: 1. In other aspects, the neutral active and N-glycosylatedhyaluronidase polypeptide has a sequence of amino acids having at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with the polypeptide or truncated form ofsequence of amino acids set forth in SEQ ID NO: 1. In yet anotheraspect, the neutral active and N-glycosylated hyaluronidase polypeptideis a full-length PH20 or is a C-terminal truncated form of the PH20,that has amino acid substitutions, whereby the hyaluronidase polypeptidehas a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with the polypeptide SEQ ID NO: 1 or the with the correspondingtruncated forms thereof. In some aspects, the hyaluronidase polypeptideused in the methods provided herein is encoded by a nucleic acidmolecule that has a sequence of nucleotides set forth in SEQ ID NO:49.In some aspects, the hyaluronidase polypeptide used in the methodsprovided herein is secreted in CHO cells. In a particular example, thePH20 is designated rHuPH20.

In one embodiment of the methods, the hyaluronidase is a neutral activesoluble hyaluronidase polypeptide containing at least one N-linked sugarmoiety. In a particular aspect, the N-linked sugar moiety is covalentlyattached to an asparagine residue of the polypeptide. In another aspect,the hyaluronidase is glycosylated at least two sites.

In some aspects, the hyaluronidase used in the methods provided herein,the soluble hyaluronidase has a sequence of amino acids included in SEQID NO:1 or a sequence that has at least about 91% amino acid sequenceidentity with a sequence of amino acids included in SEQ ID NO:1, wherebythe hyaluronidase is soluble and truncated at the C-terminus at an aminoacid residue that is between amino acid residues 467 to 483, inclusive,of SEQ ID NO:1 or corresponding residues in a polypeptide that has about91% sequence identity therewith. In a particular example, thehyaluronidase is encoded by a nucleic acid molecule that encodes aminoacids 1-482 of SEQ ID NO:1 or amino acids 36-482 of SEQ ID NO:1. Inother examples, the soluble hyaluronidase includes at least amino acidresidues 36-464 of SEQ ID NO: 1. In other examples, the hyaluronidaseincludes a sequence of amino acids set forth in SEQ ID NO:1 that istruncated at an amino acid residue that is or is between amino acidresidues 467 to 483. For example, the hyaluronidase can include asequence of amino acids set forth in SEQ ID NO:1 that is truncated at anamino acid residue selected from among amino acid residues 467, 477,478, 479, 480, 481, 482 and 483. In other examples, the hyaluronidaseincludes a sequence of amino acids set forth in SEQ ID NO:1 that istruncated at a residue selected from among amino acid residues 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 490, 491,492, 493, 494, 495, 496, 497, 498, 499 and 500. In some aspects, thehyaluronidase polypeptide used in the methods provided herein is aC-terminal truncated form produced and secreted in mammalian cells. In aparticular example, the mammalian cells are CHO cells.

In the methods provided herein, the hyaluronan degrading enzyme isPEGylated. In a particular example, the PEG moieties are branched. Insome examples, the PEG moieties are selected from among mPEG-SBA (5kDa), mPEG-SBA (20 kDa), mPEG-SBA (30 kDa), mPEG-SMB (20 kDa), mPEG-SMB(30 kDa), mPEG-butyraldehyde (30 kDa), mPEG-SPA (20 kDa), mPEG-SPA (30kDa), mPEG2-NHS (10 kDa branched), mPEG2-NHS (20 kDa branched), mPEG-NHS(40 kDa branched), mPEG2-NHS (60 kDa branched), PEG-NHS-biotin (5 kDabiotinylated), PEG-p-nitrophenyl-carbonate (30 kDa) andPEG-propionaldehyde (30 kDa).

In the methods herein, the adverse effects can be a result of treatmentby the anti-hyaluronan agent of a hyaluronan associated disease orcondition. In one example, the hyaluronan associated disease orcondition is one that is associated with high interstitial fluidpressure, a cancer, edema, disc pressure and an inflammatory disease.For example, a disease or condition that is edema can be caused by organtransplant, stroke or brain trauma. In another example, an inflammatorydisease or condition includes Rheumatoid arthritis, scleroderma,periodontitis, psoriasis, atherosclerosis, chronic wounds, Crohn'sdisease, ulcerative colitis and inflammatory bowel disease.

In a further example, the adverse effect is a result of treatment by theanti-hyaluronan agent of a cancer that is a tumor, for example a solidtumor. The tumor can be one that has increased cellular and/or stromalexpression of a hyaluronan, compared to a non-cancerous tissue of thesame tissue type or compared to a non-metastatic tumor of the sametumor-type. In one example, the cancer is a late-stage cancer, ametastatic cancer and an undifferentiated cancer. In another example,the cancer is ovarian cancer, in situ carcinoma (ISC), squamous cellcarcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lungcancer, breast cancer, brain cancer or colon cancer.

Also provided herein are uses and compositions containing acorticosteroid for ameliorating adverse effects associated withtreatment with an anti-hyaluronan agent.

Also provided herein are methods of treatment and uses of apolymer-conjugated hyaluronan-degrading enzyme (i.e. ahyaluronan-degrading enzyme modified by conjugation to a polymer) foruse in treating a hyaluronan-associated disease or condition. In suchmethods or uses, the hyaluronan-degrading enzyme is administered in adosage range or amount of between or about between 0.01 μg/kg (of thesubject) to 15 μg/kg. For example, in the methods provided herein, ahyaluronan-degrading enzyme is administered in a dosage range amount ofbetween or about between 0.05 μg/kg to 10 μg/kg, 0.75 μg/kg to 7.5 μg/kgor 1.0 μg/kg to 3.0 μg/kg. The frequency of administration is twiceweekly, once weekly, once every 14 days, once every 21 days or onceevery month. In particular examples, the hyaluronan-degrading enzyme isadministered in a dosage regime where the cycle of administration is oneweek, two weeks, three weeks or four weeks. The cycle of administrationcan be repeated a plurality of times. Also, in some examples, after acycle of administration, administration is discontinued for apredetermined period of time and then resumed in a subsequent cycle ofadministration. In some examples, the frequency of administration in thefirst cycle of administration is the same of different than thefrequency of administration in subsequent cycles of administration. In aparticular example, the hyaluronan-degrading enzyme is administeredtwice weekly in the first cycle of administration and once weekly forsubsequent cycles of administration. In such examples, the cycle ofadministration is 4 weeks.

For example, the hyaluronan-degrading enzyme is used or formulated forsingle dosage administration of an amount in a range between or aboutbetween 0.5 μg to 1450 μg or 150 Units (U) to 45,000 Units for treatinga hyaluronan-associated disease or condition. In such methods and usesthe hyaluronan-degrading enzyme can be administered as a unit dosage of0.5 μg to 1450 μg or 150 Units (U) to 45,000 Units at a frequency of atleast once a week for a cycle of at least 4 weeks. In particularexamples, single dosage administration or unit dose is of an amount in arange between or about between 0.75 μg to 1125 μg; 3.75 mg to 750 μg; 56μg to 565 μg; or 75 μg to 225 μg. In other examples, the single dosageadministration or unit dose is in a range between or about between 24Units (U) to 36,000 U; 120 U to 24,000 U; 1500 U to 18,000 U; or 2400 Uto 7200 U.

In any of the methods or uses herein, in particular for treating ahyaluronan-associated disease or condition, hyaluronan expression in asample from the subject is measured prior to treatment. The hyaluronanassociated disease or condition is associated with high interstitialfluid pressure, a cancer, edema, disc pressure or an inflammatorydisease. For example, the disease or condition can be an edema caused byorgan transplant, stroke or brain trauma. In other examples, the diseaseor condition is an inflammatory disease that is Rheumatoid arthritis,scleroderma, periodontitis, psoriasis, atherosclerosis, chronic wounds,Crohn's disease, ulcerative colitis or inflammatory bowel disease. Inparticular examples, the disease or condition is cancer and the canceris a tumor. For example, the disease or condition is a solid tumor. Thetumor can be one that has increased cellular and/or stromal expressionof a hyaluronan, compared to a non-cancerous tissue of the same tissuetype or compared to a non-metastatic tumor of the same tumor-type. Wherethe disease or condition is a cancer, the cancer is a late-stage cancer,a metastatic cancer or an undifferentiated cancer. For example, thecancer is ovarian cancer, in situ carcinoma (ISC), squamous cellcarcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lungcancer, breast cancer, brain cancer or colon cancer.

In some examples, a corticosteroid can further be administered. Forexample, a corticosteroid is a glucocorticoid. For example, thecorticosteroid is a glucocorticoid that is a cortisone, dexamethasone,hydrocortisone, methylprednisolone, prednisolone or prednisone. In aparticular example, the glucocorticoid is a dexamethasone.

In some embodiments of the methods provided herein, the methods furtherinvolve a step of administering a second agent or treatment. In someaspects, the second agent or treatment is a cancer treatment, such as,for example, surgery, radiation, a chemotherapeutic agent, a biologicalagent, a polypeptide, an antibody, a peptide, a small molecule, a genetherapy vector, a virus or DNA. In particular examples, the second agentis a cancer treatment selected from among Acivicins; Aclarubicins;Acodazoles; Acronines; Adozelesins; Aldesleukins; Alemtuzumabs;Alitretinoins (9-Cis-Retinoic Acids); Allopurinols; Altretamines;Alvocidibs; Ambazones; Ambomycins; Ametantrones; Amifostines;Aminoglutethimides; Amsacrines; Anastrozoles; Anaxirones; Ancitabines;Anthramycins; Apaziquones; Argimesnas; Arsenic Trioxides; Asparaginases;Asperlins; Atrimustines; Azacitidines; Azetepas; Azotomycins;Banoxantrones; Batabulins; Batimastats; BCG Live; Benaxibines;Bendamustines; Benzodepas; Bexarotenes; Bevacizumab; Bicalutamides;Bietaserpines; Biricodars; Bisantrenes; Bisantrenes; BisnafideDimesylates; Bizelesins; Bleomycins; Bortezomibs; Brequinars;Bropirimines; Budotitanes; Busulfans; Cactinomycins; Calusterones;Canertinibs; Capecitabines; Caracemides; Carbetimers; Carboplatins;Carboquones; Carmofurs; Carmustines with Polifeprosans; Carmustines;Carubicins; Carzelesins; Cedefingols; Celecoxibs; Cemadotins;Chlorambucils; Cioteronels; Cirolemycins; Cisplatins; Cladribines;Clanfenurs; Clofarabines; Crisnatols; Cyclophosphamides; Cytarabineliposomals; Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;Decitabines; Denileukin Diftitoxes; Dexniguldipines; Dexonnaplatins;Dexrazoxanes; Dezaguanines; Diaziquones; Dibrospidiums; Dienogests;Dinalins; Disermolides; Docetaxels; Dofequidars; Doxifluridines;Doxorubicin liposomals; Doxorubicin HCL; Doxorubicin HCL liposomeinjection; Doxorubicins; Droloxifenes; Dromostanolone Propionates;Duazomycins; Ecomustines; Edatrexates; Edotecarins; Eflomithines;Elacridars; Elinafides; Elliott's B Solutions; Elsamitrucins; Emitefurs;Enloplatins; Enpromates; Enzastaurins; Epipropidines; Epirubicins;Epoetin alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles; Fazarabines;Fenretinides; Filgrastims; Floxuridines; Fludarabines; Fluorouracils;5-fluorouracils; Fluoxymesterones; Fluorocitabines; Fosquidones;Fostriecins; Fostriecins; Fotretamines; Fulvestrants; Galarubicins;Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins; Geroquinols;Gimatecans; Gimeracils; Gloxazones; Glufosfamides; Goserelin acetates;Hydroxyureas; Ibritumomabs/Tiuxetans; Idarubicins; Ifosfamides;Ilmofosines; Ilomastats; Imatinib mesylates; Imexons; Improsulfans;Indisulams; Inproquones; Interferon alfa-2 as; Interferon alfa-2bs;Interferon Alfas; Interferon Betas; Interferon Gammas; Interferons;Interleukin-2s and other Interleukins (including recombinantInterleukins); Intoplicines; Iobenguanes [131-I]; Iproplatins;Irinotecans; Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines;Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins;Leuprolides; Leuprorelins (Leuprorelides); Levamisoles; Lexacalcitols;Liarozoles; Lobaplatins; Lometrexols; Lomustines/CCNUs; Lomustines;Lonafarnibs; Losoxantrones; Lurtotecans; Mafosfamides; Mannosulfans;Marimastats; Masoprocols; Maytansines; Mechlorethamines;Mechlorethamines/Nitrogen mustards; Megestrol acetates; Megestrols;Melengestrols; Melphalans; MelphalanslL-PAMs; Menogarils; Mepitiostanes;Mercaptopurines; 6-Mercaptopurine; Mesnas; Metesinds; Methotrexates;Methoxsalens; Metomidates; Metoprines; Meturedepas; Miboplatins;Miproxifenes; Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone Phenpropionates;Nedaplatins; Nelzarabines; Nemorubicins; Nitracrines; Nocodazoles;Nofetumomabs; Nogalamycins; Nolatrexeds; Nortopixantrones; Octreotides;Oprelvekins; Ormaplatins; Ortataxels; Oteracils; Oxaliplatins;Oxisurans; Oxophenarsines; Paclitaxels; Pamidronates; Patubilones;Pegademases; Pegaspargases; Pegfilgrastims; Peldesines; Peliomycins;Pelitrexols; Pemetrexeds; Pentamustines; Pentostatins; Peplomycins;Perfosfamides; Perifosines; Picoplatins; Pinafides; Pipobromans;Piposulfans; Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds;Plicamycid Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimersodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins; Sabarubicins;Safingols; Sargramostims; Satraplatins; Sebriplatins; Semustines;Simtrazenes; Sizofurans; Sobuzoxanes; Sorafenibs; Sparfosates; SparfosicAcids; Sparsomycins; Spirogermaniums; Spiromustines; Spiroplatins;Spiroplatins; Squalamines; Streptonigrins; Streptovarycins;Streptozocins; Sufosfamides; Sulofenurs; Sunitinib Malate; 6-thioguanine(6-TG); Tacedinalines; Talcs; Talisomycins; Tallimustines; Tamoxifens;Tariquidars; Tauromustines; Tecogalans; Tegafurs; Teloxantrones;Temoporfins; Temozolomides; Teniposides/VM-26s; Teniposides;Teroxirones; Testolactones; Thiamiprines; Thioguanines; Thiotepas;Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;Tirapazamines; Topixantrones; Topotecans; Toremifenes; Tositumomabs;Trabectedins (Ecteinascidin 743); Trastuzumabs; Trestolones;Tretinoins/ATRA; Triciribines; Trilostanes; Trimetrexates; TriplatinTetranitrates; Triptorelins; Trofosfamides; Tubulozoles; Ubenimexs;Uracil Mustards; Uredepas; Valrubicins; Valspodars; Vapreotides;Verteporfins; Vinblastines; Vincristines; Vindesines; Vinepidines;Vinflunines; Vinformides; Vinglycinates; Vinleucinols; Vinleurosines;Vinorelbines; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles;Xanthomycin As (Guamecyclines); Zeniplatins; Zilascorbs [2-H];Zinostatins; Zoledronate; Zorubicins; and Zosuquidars.

In the methods provided herein, the subject administered the PEGylatedhyaluronan degrading enzyme is a human subject. In a particular aspectof the methods, the subject has cancer and treatment comprisessystemically administering a PEGylated hyaluronan degrading enzyme.

In any of the methods or uses provided herein using ahyaluronan-degrading enzyme conjugated to a polymer, the specificactivity can be at least or about 20,000 U/mg, 25,000 U/mg, 30,000 U/mg,31,000 U/mg, 32,000 U/mg, 33,000 U/mg, 34,000 U/mg, 35,000 U/mg, 36,000U/mg, 37,000 U/mg, 38,000 U/mg, 39,000 U/mg, 40,000 U/mg, 45,000 U/mg,50,000 U/mg, 55,000 U/mg, 60,000 U/mg or more.

Further provided herein is a combination containing a first compositioncontaining a hyaluronan degrading enzyme formulated for intravenousadministration and a second composition comprising a corticosteroid. Inparticular, in the combinations provided herein the hyaluronan degradingenzyme is a PEGylated hyaluronan degrading enzyme, such as a PEGylatedPH20 (e.g. PEGPH20). In other examples of the combinations providedherein, the corticosteroid is a glucocorticoid such as dexamethasone.

Also provided herein is a device containing a composition containing ahyaluronan-degrading enzyme formulated in an amount for directadministration in a range between or about between 0.5 μg to 1450 μg or150 Units (U) to 45,000 Units per dose. The hyaluronan-degrading enzymecontained in the device is modified by conjugation to a polymer, and thedevice is for dispensing the composition and is visibly or detectablymarked to indicate a single dosage amount for administration. In someexamples, the device is for dispensing a plurality of single doses, andeach mark indicates a single dosage. The device can be a syringe or atube or vial. In some examples, the device is a packaged as a kit orcombination and contains a composition also contains a corticosteroidformulated for single dosage administration.

Any of the hyaluronan-degrading enzymes described herein or known in theart can be used in the methods or uses for ameliorating a side effect,the methods or uses for treating a hyaluronan-associated disease orcondition or in the combinations or contained in the devices herein.Exemplary hyaluronan degrading enzymes provided herein, includingsoluble hyaluronidases and preparations thereof are described, forexample, in U.S. Publication Nos. US20040268425, US20050260186,US20060104968, US20090123367, US20090181013, US20090181032,US20090214505, US20090253175 and US20100143457 and U.S. Pat. No.7,767,429.

DETAILED DESCRIPTION Outline

-   -   A. DEFINITIONS    -   B. OVERVIEW        -   1. Anti-Hyaluronan Agents and Hyaluronan-associated            diseases, conditions and/or disorders        -   2. Adverse effects associated with treatment with            anti-hyaluronan agents        -   3. Use of corticosteroids to ameliorate the adverse effects            of anti-Hyaluronan Agents    -   C. ANTI-HYALURONAN AGENTS        -   1. Agents that Inhibit Hyaluronan Synthesis        -   2. Hyaluronan Degrading Enzymes            -   a. Hyaluronidases                -   i. Mammalian-type hyaluronidases PH20                -   ii. Bacterial hyaluronidases                -   iii. Hyaluronidases from leeches, other parasites                    and crustaceans            -   b. Other hyaluronan degrading enzymes            -   c. Soluble hyaluronan degrading enzymes                -   i. Soluble Human PH20                -   ii. rHuPH20            -   d. Glycosylation of hyaluronan degrading enzymes            -   e. Modified (Polymer-Conjugated) hyaluronan degrading                enzymes                -   PEGylated Soluble hyaluronan degrading enzymes    -   D. METHODS OF PRODUCING NUCLEIC ACIDS AND ENCODED POLYPEPTIDES        OF HYALURONAN DEGRADING ENZYMES        -   1. Vectors and Cells        -   2. Expression            -   a. Prokaryotic Cells            -   b. Yeast Cells            -   c. Insect Cells            -   d. Mammalian Cells            -   e. Plants        -   3. Purification Techniques        -   4. PEGylation of Hyaluronan degrading enzymes polypeptides    -   E. CORTICOSTEROIDS    -   F. USE OF CORTICOSTEROIDS TO AMELIORATE THE ADVERSE EFFECTS OF        AN ANTI-HYALURONAN AGENT        -   1. Pharmaceutical Compositions and Formulations        -   2. Dosages and Administration            -   a. Corticosteroid            -   b. Anti-Hyaluronan Agent                -   i. Leflunomide and Derivatives                -   ii. Hyaluronan Degrading Enzyme        -   3. Combination Treatment            -   Anti-Cancer Agents and Other Treatments        -   4. Packaging and Articles of Manufacture    -   G. METHODS OF ASSESSING ACTIVITY AND EFFECTS OF ANTI-HYALURONAN        AGENTS AND CORTICOSTEROIDS        -   1. Methods to Assess Side Effects        -   2. Anti-Hyaluronan Activity            -   a. Assays to Assess the Activity of a Hyaluronan                Degrading Enzyme            -   b. Assays in Animal Models            -   c. Assays in Humans            -   d. Pharmacokinetics    -   H. USE OF ANTI-HYALURONAN AGENTS IN TREATING        HYALURONAN-ASSOCIATED CONDITIONS, DISEASES AND DISORDERS        -   1. Selection of Subjects for Treatment And Assessing            Treatment Effects            -   a. Assays for detection of Hyaluronan-Associated Disease                Markers            -   b. Detection of Hyaluronan-Associated Markers Relative                to Control Samples        -   2. Use in Treating Cancers    -   I. EXAMPLES

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belongs/belong. All patents, patentapplications, published applications and publications, Genbanksequences, databases, websites and other published materials referred tothroughout the entire disclosure herein, unless noted otherwise, areincorporated by reference in their entirety. In the event that there area plurality of definitions for terms herein, those in this sectionprevail. Where reference is made to a URL or other such identifier oraddress, it understood that such identifiers can change and particularinformation on the internet can come and go, but equivalent informationcan be found by searching the internet. Reference thereto evidences theavailability and public dissemination of such information.

As used herein, “adverse effect” or “side effect” refers to a harmful,deleterious and/or undesired effect of administering an anti-hyaluronanagent, such as a PEGylated hyaluronan degrading enzyme, such as PEGPH20to a subject. Exemplary of side effects are musculoskeletal sideeffects. Side effects or adverse effects are graded on toxicity andvarious toxicity scales exist providing definitions for each grade.Exemplary of such scales are toxicity scales of the National CancerInstitute Common Toxicity Criteria version 2.0, the World HealthOrganization or Common Terminology Criteria for Adverse Events (CTCAE)scale. Generally, the scale is as follows: Grade 1=mild side effects;Grade 2=moderate side effects; Grade 3=Severe side effects; Grade 4=LifeThreatening or Disabling side-effects; Grade 5=Fatal. Assigning gradesof severity is within the experience of a physician or other health careprofessional.

As used herein, a dose-limiting toxicity (DLT) refers to the dose of adrug that produces side effects severe enough to prevent larger dosesbeing given. It is within the level of skill of a skilled physician toassign or determine a DLT depending on the treatment protocol, thedisease to be treated, the dosage regime and the particular patient tobe treated. An exemplary definition of a DLT for treatment with ananti-hyaluronan agent treatment (in the absence of a corticosteroid orin its presence) is the dose in which an adverse event or side effect isobserved that is related to the anti-hyaluronan treatment and that isdefined on a toxicity scale of at least Grade 3 or higher and is anon-hematologic toxicity, or an ongoing or persistent Grade 2 toxicitythat fails to resolve over the course of treatment and that limits thepatient's ability to comply with the protocol therapy, or any Grade 4 orprolonged Grade 3 hematological toxicity. DLT's do not include nausea orvomiting that occurred without prophylactic anti-emetic therapy and thatcan be effectively treated with such therapy, or side effects thatresolve on their own within a few hours to 24 hours.

As used herein, “musculoskeletal effect” or “musculoskeletal sideeffect” refers to effects on the system of muscles, tendons, ligaments,bones, joints and associated tissues. Musculoskeletal side effectsinclude muscle and joint pain, stiffness of upper and lower extremities,cramping, myositis, muscle soreness and tenderness over the entire body,weakness, fatigue and a decrease in range of motion at knee and elbowjoints. It is within the level of a skilled physician to assign gradesof severity of observed or measured musculoskeletal side effects basedon toxicity scales. It also is within the level of a skilled physicianto assign a DLT to an observed musculoskeletal side effect.

As used herein, “ameliorating” or “reducing” a side effect or adverseevent, or variations thereof, refers to lessening adverse effects orside effects, whether permanent or temporary, lasting or transient. Forpurposes herein, a side effect of an administered anti-hyaluronan agent,such as a musculoskeletal side effect, is deemed ameliorated by acorticosteroid when there is a reduction or lessening in the grade ofseverity measured on a toxicity scale for the side effect in thepresence of the corticosteroid compared to in its absence. In oneexample, a side effect is ameliorated when the observed or measuredtoxicity of an administered anti-hyaluronan agent (observed followingsingle dosage administration, multiple dosage administration or byvirtue of the dosage regime) of Grade 3 or higher is reduced to a Grade1 or Grade 2 in the presence of a corticosteroid. In another example, aside effect is ameliorated when the DLT of an administeredanti-hyaluronan agent is eliminated or increased followingadministration of the corticosteroid. For example, a side effect isameliorated when a DLT of 0.05 mg/mL from an administeredanti-hyaluronan agent is eliminated or increased by administration of acorticosteroid to the subject, such that the same dose or higher dose ofanti-hyaluronan agent can be administered with reduced side effect orwhen the DLT is increased to greater than 0.05 mg/mL, such as 0.5 mg/mL.

As used herein, “intravenous administration” refers to delivery of atherapeutic directly into a vein.

As used herein, an anti-hyaluronan agent refers to any agent thatmodulates hyaluronan (HA) synthesis or degradation, thereby alteringhyaluronan levels in a tissue or cell. For purposes herein,anti-hyaluronan agents reduce hyaluronan levels in a tissue or cellcompared to the absence of the agent. Such agents include compounds thatmodulate the expression of genetic material encoding HA synthase (HAS)and other enzymes or receptors involved in hyaluronan metabolism, orthat modulate the proteins that synthesize or degrade hyaluronanincluding HAS function or activity. The agents include small-molecules,nucleic acids, peptides, proteins or other compounds. For example,anti-hyaluronan agents include, but are not limited to, antisense orsense molecules, antibodies, enzymes, small molecule inhibitors and HASsubstrate analogs.

As used herein, a hyaluronan degrading enzyme refers to an enzyme thatcatalyzes the cleavage of a hyaluronan polymer (also referred to ashyaluronic acid or HA) into smaller molecular weight fragments.Exemplary of hyaluronan degrading enzymes are hyaluronidases, andparticular chondroitinases and lyases that have the ability todepolymerize hyaluronan. Exemplary chondroitinases that are hyaluronandegrading enzymes include, but are not limited to, chondroitin ABC lyase(also known as chondroitinase ABC), chondroitin AC lyase (also known aschondroitin sulfate lyase or chondroitin sulfate eliminase) andchondroitin C lyase. Chondroitin ABC lyase comprises two enzymes,chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) andchondroitin-sulfate-ABC exolyase (EC 4.2.2.21). An exemplarychondroitin-sulfate-ABC endolyases and chondroitin-sulfate-ABC exolyasesinclude, but are not limited to, those from Proteus vulgaris andFlavobacterium heparinum (the Proteus vulgaris chondroitin-sulfate-ABCendolyase is set forth in SEQ ID NO:98; Sato et al. (1994) Appl.Microbiol. Biotechnol. 41(1):39-46). Exemplary chondroitinase AC enzymesfrom the bacteria include, but are not limited to, those fromFlavobacterium heparinum Victivallis vadensis, set forth in SEQ IDNO:99, and Arthrobacter aurescens (Tkalec et al. (2000) Applied andEnvironmental Microbiology 66(1):29-35; Ernst et al. (1995) CriticalReviews in Biochemistry and Molecular Biology 30(5):387-444). Exemplarychondroitinase C enzymes from the bacteria include, but are not limitedto, those from Streptococcus and Flavobacterium (Hibi et al. (1989)FEMS-Microbiol-Lett. 48(2):121-4; Michelacci et al. (1976) J. Biol.Chem. 251:1154-8; Tsuda et al. (1999) Eur. J. Biochem. 262:127-133).

As used herein, hyaluronidase refers to a class of hyaluronan degradingenzymes. Hyaluronidases include bacterial hyaluronidases (EC 4.2.2.1 orEC 4.2.99.1), hyaluronidases from leeches, other parasites, andcrustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC3.2.1.35). Hyaluronidases include any of non-human origin including, butnot limited to, murine, canine, feline, leporine, avian, bovine, ovine,porcine, equine, piscine, ranine, bacterial, and any from leeches, otherparasites, and crustaceans. Exemplary non-human hyaluronidases include,hyaluronidases from cows (SEQ ID NOS:10, 11, 64 and BH55 (U.S. Pat. Nos.5,747,027 and 5,827,721), yellow jacket wasp (SEQ ID NOS:12 and 13),honey bee (SEQ ID NO:14), white-face hornet (SEQ ID NO:15), paper wasp(SEQ ID NO:16), mouse (SEQ ID NOS:17-19, 32), pig (SEQ ID NOS:20-21),rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25), sheep (SEQ ID NOS:26,27, 63 and 65), chimpanzee (SEQ ID NO:101), Rhesus monkey (SEQ IDNO:102), orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ ID NO:29),guinea pig (SEQ ID NO:30), Arthrobacter sp. (strain FB24) (SEQ IDNO:67), Bdellovibrio bacteriovorus (SEQ ID NO:68), Propionibacteriumacnes (SEQ ID NO:69), Streptococcus agalactiae ((SEQ ID NO:70); 18RS21(SEQ ID NO:71); serotype Ia (SEQ ID NO:72); serotype III (SEQ ID NO:73),Staphylococcus aureus (strain COL (SEQ ID NO:74); strain MRSA252 (SEQ IDNOS:75 and 76); strain MSSA476 (SEQ ID NO:77); strain NCTC 8325 (SEQ IDNO:78); strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300 (SEQID NO:81), Streptococcus pneumoniae ((SEQ ID NO:82); strain ATCCBAA-255/R6 (SEQ ID NO:83); serotype 2, strain D39/NCTC 7466 (SEQ IDNO:84), Streptococcus pyogenes (serotype M1) (SEQ ID NO:85); serotypeM2, strain MGAS10270 (SEQ ID NO:86); serotype M4, strain MGAS10750 (SEQID NO:87); serotype M6 (SEQ ID NO:88); serotype M12, strain MGAS2096(SEQ ID NOS:89 and 90); serotype M12, strain MGAS9429 (SEQ ID NO:91);serotype M28 (SEQ ID NO:92); Streptococcus suis (SEQ ID NOS:93-95);Vibrio fischeri (strain ATCC 700601/ES114 (SEQ ID NO:96)), and theStreptomyces hyaluronolyticus hyaluronidase enzyme, which is specificfor hyaluronic acid and does not cleave chondroitin or chondroitinsulfate (Ohya, T. and Kaneko, Y. (1970) Biochim. Biophys. Acta 198:607).Hyaluronidases also include those of human origin. Exemplary humanhyaluronidases include HYAL1 (SEQ ID NO:36), HYAL2 (SEQ ID NO:37), HYAL3(SEQ ID NO:38), HYAL4 (SEQ ID NO:39), and PH20 (SEQ ID NO:1). Alsoincluded amongst hyaluronidases are soluble hyaluronidases, including,ovine and bovine PH20, soluble human PH20 and soluble rHuPH20. Examplesof commercially available bovine or ovine soluble hyaluronidases includeVitrase® (ovine hyaluronidase), Amphadase® (bovine hyaluronidase) andHydase™ (bovine hyaluronidase).

As used herein, “purified bovine testicular hyaluronidase” refers to abovine hyaluronidase purified from bovine testicular extracts (see U.S.Pat. Nos. 2,488,564, 2,488,565, 2,806,815, 2,808,362, 2,676,139,2,795,529, 5,747,027 and 5,827,721). Examples of commercially availablepurified bovine testicular hyaluronidases include Amphadase® andHydase™, and bovine hyaluronidases, including, but not limited to, thoseavailable from Sigma Aldrich, Abnova, EMD Chemicals, GenWay Biotech,Inc., Raybiotech, Inc., and Calzyme. Also included are recombinantlyproduced bovine hyaluronidases, such as but not limited to, thosegenerated by expression of a nucleic acid molecule set forth in any ofSEQ ID NOS:190-192.

As used herein, “purified ovine testicular hyaluronidase” refers to anovine hyaluronidase purified from ovine testicular extracts (see U.S.Pat. Nos. 2,488,564, 2,488,565 and 2,806,815 and International PCTApplication No. WO2005/118799). Examples of commercially availablepurified ovine testicular extract include Vitrase®, and ovinehyaluronidases, including, but not limited to, those available fromSigma Aldrich, Cell Sciences, EMD Chemicals, GenWay Biotech, Inc.,Mybiosource.com and Raybiotech, Inc. Also included are recombinantlyproduced ovine hyaluronidases, such as, but not limited to, thosegenerated by expression of a nucleic acid molecule set forth in any ofSEQ ID NOS:66 and 193-194.

As used herein, “PH20” refers to a type of hyaluronidase that occurs insperm and is neutral-active. PH-20 occurs on the sperm surface, and inthe lysosome-derived acrosome, where it is bound to the inner acrosomalmembrane. PH20 includes those of any origin including, but not limitedto, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, murine, bovine,ovine, guinea pig, rabbit and rat origin. Exemplary PH20 polypeptidesinclude those from human (SEQ ID NO:1), chimpanzee (SEQ ID NO:101),Rhesus monkey (SEQ ID NO:102), Cynomolgus monkey (SEQ ID NO:29), cow(e.g., SEQ ID NOS:11 and 64), mouse (SEQ ID NO:32), rat (SEQ ID NO:31),rabbit (SEQ ID NO:25), sheep (SEQ ID NOS:27, 63 and 65) and guinea pig(SEQ ID NO:30).

Reference to hyaluronan degrading enzymes includes precursor hyaluronandegrading enzyme polypeptides and mature hyaluronan degrading enzymepolypeptides (such as those in which a signal sequence has beenremoved), truncated forms thereof that have activity, and includesallelic variants and species variants, variants encoded by splicevariants, and other variants, including polypeptides that have at least40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more sequence identity to the precursor polypeptides set forth inSEQ ID NOS: 1 and 10-48, 63-65, 67-102, or the mature forms thereof. Forexample, reference to hyaluronan degrading enzyme also includes thehuman PH20 precursor polypeptide variants set forth in SEQ ID NOS:50-51.Hyaluronan degrading enzymes also include those that contain chemical orposttranslational modifications and those that do not contain chemicalor posttranslational modifications. Such modifications include, but arenot limited to, PEGylation, albumination, glycosylation, farnesylation,carboxylation, hydroxylation, phosphorylation, and other polypeptidemodifications known in the art. A truncated PH20 hyaluronidase is anyC-terminal shortened form thereof, particularly forms that are truncatedand neutral active when N-glycosylated.

As used herein, a “soluble PH20” refers to any form of PH20 that issoluble under physiologic conditions. A soluble PH20 can be identified,for example, by its partitioning into the aqueous phase of a Triton®X-114 solution at 37° C. (Bordier et al., (1981) J. Biol. Chem.,256:1604-7). Membrane-anchored PH20, such as lipid-anchored PH20,including GPI-anchored PH20, will partition into the detergent-richphase, but will partition into the detergent-poor or aqueous phasefollowing treatment with Phospholipase-C. Included among soluble PH20are membrane-anchored PH20 in which one or more regions associated withanchoring of the PH20 to the membrane has been removed or modified,where the soluble form retains hyaluronidase activity. Soluble PH20 alsoincludes recombinant soluble PH20 and those contained in or purifiedfrom natural sources, such as, for example, testes extracts from sheepor cows. Exemplary of such soluble PH20 is soluble human PH20.

As used herein, soluble human PH20 or sHuPH20 includes PH20 polypeptideslacking all or a portion of the glycosylphosphatidylinositol (GPI)anchor sequence at the C-terminus such that upon expression, thepolypeptides are soluble under physiological conditions. Solubility canbe assessed by any suitable method that demonstrates solubility underphysiologic conditions. Exemplary of such methods is the Triton® X-114assay, that assesses partitioning into the aqueous phase and that isdescribed above and in the examples. In addition, a soluble human PH20polypeptide is, if produced in CHO cells, such as CHO-S cells, apolypeptide that is expressed and is secreted into the cell culturemedium. Soluble human PH20 polypeptides, however, are not limited tothose produced in CHO cells, but can be produced in any cell or by anymethod, including recombinant expression and polypeptide synthesis.Reference to secretion by CHO cells is definitional. Hence, if apolypeptide could be expressed and secreted by CHO cells and is soluble,i.e. partitions into the aqueous phase when extracted with Triton®X-114, it is a soluble PH20 polypeptide whether or not it isso-produced. The precursor polypeptides for sHuPH20 polypeptides caninclude a signal sequence, such as a heterologous or non-heterologous(i.e. native) signal sequence. Exemplary of the precursors are thosethat include a signal sequence, such as the native 35 amino acid signalsequence at amino acid positions 1-35 (see, e.g., amino acids 1-35 ofSEQ ID NO:1).

As used herein, an “extended soluble PH20” or “esPH20” includes solublePH20 polypeptides that contain residues up to the GPI anchor-attachmentsignal sequence and one or more contiguous residues from the GPI-anchorattachment signal sequence such that the esPH20 is soluble underphysiological conditions. Solubility under physiological conditions canbe determined by any method known to those of skill in the art. Forexample, it can be assessed by the Triton® X-114 assay described aboveand in the examples. In addition, as discussed above, a soluble PH20 is,if produced in CHO cells, such as CHO-S cells, a polypeptide that isexpressed and is secreted into the cell culture medium. Soluble humanPH20 polypeptides, however, are not limited to those produced in CHOcells, but can be produced in any cell or by any method, includingrecombinant expression and polypeptide synthesis. Reference to secretionby CHO cells is definitional. Hence, if a polypeptide could be expressedand secreted by CHO cells and is soluble, i.e. partitions into theaqueous phase when extracted with Triton® X-114, it is a soluble PH20polypeptide whether or not it is so-produced. Human soluble esPH20polypeptides include, in addition to residues 36-490, one or morecontiguous amino acids from amino acid residue position 491 of SEQ IDNO:1, inclusive, such that the resulting polypeptide is soluble.Exemplary human esPH20 soluble polypeptides are those that have aminoacids residues corresponding to amino acids 36-491, 36-492, 36-493,36-494, 36-495, 36-496 and 36-497 of SEQ ID NO:1. Exemplary of these arethose with an amino acid sequence set forth in any of SEQ ID NOS:151-154and 185-187. Also included are allelic variants and other variants, suchas any with 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with thecorresponding polypeptides of SEQ ID NOS:151-154 and 185-187 that retainneutral activity and are soluble. Reference to sequence identity refersto variants with amino acid substitutions.

As used herein, reference to “esPH20s” includes precursor esPH20polypeptides and mature esPH20 polypeptides (such as those in which asignal sequence has been removed), truncated forms thereof that haveenzymatic activity (retaining at least 1%, 10%, 20%, 30%, 40%, 50% ormore of the full-length form) and are soluble, and includes allelicvariants and species variants, variants encoded by splice variants, andother variants, including polypeptides that have at least 40%, 45%, 50%,55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the precursor polypeptides setforth in SEQ ID NOS:1 and 3, or the mature forms thereof.

As used herein, reference to “esPH20s” also include those that containchemical or posttranslational modifications and those that do notcontain chemical or posttranslational modifications. Such modificationsinclude, but are not limited to, PEGylation, albumination,glycosylation, farnesylation, carboxylation, hydroxylation,phosphorylation, and other polypeptide modifications known in the art.

As used herein, “soluble recombinant human PH20 (rHuPH20)” refers to acomposition containing solubles form of human PH20 as recombinantlyexpressed and secreted in Chinese Hamster Ovary (CHO) cells. SolublerHuPH20 is encoded by nucleic acid molecule that includes the signalsequence and is set forth in SEQ ID NO:49. The nucleic acid encodingsoluble rHuPH20 is expressed in CHO cells which secrete the maturepolypeptide. As produced in the culture medium, there is heterogeneityat the C-terminus so that the product includes a mixture of species thatcan include any one or more of SEQ ID NO:4 to SEQ ID NO:9 in variousabundance.

Similarly, for other forms of PH20, such as the esPH20s, recombinantlyexpressed polypeptides and compositions thereof can include a pluralityof species whose C-terminus exhibits heterogeneity. For example,compositions of recombinantly expressed esPH20 produced by expression ofthe polypeptide of SEQ ID NO:107, which encodes an esPH20 that has aminoacids 36-497, can include forms with fewer amino acids, such as 36-496,36-495.

As used herein, an “N-linked moiety” refers to an asparagine (N) aminoacid residue of a polypeptide that is capable of being glycosylated bypost-translational modification of a polypeptide. Exemplary N-linkedmoieties of human PH20 include amino acids N82, N166, N235, N254, N368and N393 of human PH20 set forth in SEQ ID NO:1.

As used herein, an “N-glycosylated polypeptide” refers to a PH20polypeptide or truncated form thereto containing oligosaccharide linkageof at least three N-linked amino acid residues, for example, N-linkedmoieties corresponding to amino acid residues N235, N368 and N393 of SEQID NO:1. An N-glycosylated polypeptide can include a polypeptide wherethree, four, five and up to all of the N-linked moieties are linked toan oligosaccharide. The N-linked oligosaccharides can includeoligomannose, complex, hybrid or sulfated oligosaccharides, or otheroligosaccharides and monosaccharides.

As used herein, an “N-partially glycosylated polypeptide” refers to apolypeptide that minimally contains an N-acetylglucosamine glycan linkedto at least three N-linked moieties. A partially glycosylatedpolypeptide can include various glycan forms, including monosaccharides,oligosaccharides, and branched sugar forms, including those formed bytreatment of a polypeptide with EndoH, EndoF1, EndoF2 and/or EndoF3.

As used herein, a “deglycosylated PH20 polypeptide” refers to a PH20polypeptide in which fewer than all possible glycosylation sites areglycosylated. Deglycosylation can be effected, for example, by removingglycosylation, by preventing it, or by modifying the polypeptide toeliminate a glycosylation site. Particular N-glycosylation sites are notrequired for activity, whereas others are.

As used herein, “PEGylated” refers to covalent or other stableattachment of polymeric molecules, such as polyethylene glycol(PEGylation moiety PEG) to hyaluronan degrading enzymes, such ashyaluronidases, typically to increase half-life of the hyaluronandegrading enzyme.

As used herein, a “conjugate” refers to a polypeptide linked directly orindirectly to one or more other polypeptides or chemical moieties. Suchconjugates include fusion proteins, those produced by chemicalconjugates and those produced by any other methods. For example, aconjugate refers to soluble PH20 polypeptides linked directly orindirectly to one or more other polypeptides or chemical moieties,whereby at least one soluble PH20 polypeptide is linked, directly orindirectly to another polypeptide or chemical moiety so long as theconjugate retains hyaluronidase activity.

As used herein, a “fusion” protein refers to a polypeptide encoded by anucleic acid sequence containing a coding sequence from one nucleic acidmolecule and the coding sequence from another nucleic acid molecule inwhich the coding sequences are in the same reading frame such that whenthe fusion construct is transcribed and translated in a host cell, theprotein is produced containing the two proteins. The two molecules canbe adjacent in the construct or separated by a linker polypeptide thatcontains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6amino acids. The protein product encoded by a fusion construct isreferred to as a fusion polypeptide.

As used herein, a “polymer” refers to any high molecular weight naturalor synthetic moiety that is conjugated to, i.e. stably linked directlyor indirectly via a linker, to a polypeptide. Such polymers, typicallyincrease serum half-life, and include, but are not limited to sialicmoieties, PEGylation moieties, dextran, and sugar and other moieties,such as glycosylation. For example, hyaluronidases, such as a solublePH20 or rHuPH20, can be conjugated to a polymer.

As used herein, a hyaluronidase substrate refers to a substrate (e.g.protein or polysaccharide) that is cleaved and/or depolymerized by ahyaluronidase enzyme. Generally, a hyaluronidase substrate is aglycosaminoglycan. An exemplary hyaluronidase substrate is hyaluronan(HA).

As used herein, a hyaluronan-associated disease, disorder or conditionrefers to any disease or condition in which hyaluronan levels areelevated as cause, consequence or otherwise observed in the disease orcondition. Hyaluronan-associated diseases and conditions are associatedwith elevated hyaluronan expression in a tissue or cell, increasedinterstitial fluid pressure, decreased vascular volume, and/or increasedwater content in a tissue. Hyaluronan-associated diseases, disorders orconditions can be treated by administration of a composition containingan anti-hyaluronan agent, such as a hyaluronan degrading enzyme, such asa hyaluronidase, for example, a soluble hyaluronidase, either alone orin combination with or in addition to another treatment and/or agent.Exemplary diseases and conditions, include, but are not limited to,inflammatory diseases and hyaluronan-rich cancers. Hyaluronan richcancers include, for example, tumors, including solid tumors such aslate-stage cancers, a metastatic cancers, undifferentiated cancers,ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC),prostate cancer, pancreatic cancer, non-small cell lung cancer, breastcancer, colon cancer and other cancers. Also exemplary ofhyaluronan-associated diseases and conditions are diseases that areassociated with elevated interstitial fluid pressure, such as diseasesassociated with disc pressure, and edema, for example, edema caused byorgan transplant, stroke, brain trauma or other injury. Exemplaryhyaluronan-associated diseases and conditions include diseases andconditions associated with elevated interstitial fluid pressure,decreased vascular volume, and/or increased water content in a tissue,including cancers, disc pressure and edema. In one example, treatment ofthe hyaluronan-associated condition, disease or disorder includesamelioration, reduction, or other beneficial effect on one or more ofincreased interstitial fluid pressure (IFP), decreased vascular volume,and increased water content in a tissue.

As used herein, elevated hyaluronan levels refers to amounts ofhyaluronan in a particular tissue, body fluid or cell, dependent uponthe disease or condition. For example, as a consequence of the presenceof a hyaluronan-rich tumor, hyaluronan (HA) levels can be elevated inbody fluids, such as blood, urine, saliva and serum, and/or in thetumorous tissue or cell. The level can be compared to a standard orother suitable control, such as a comparable sample from a subject whodoes not have the HA-associated disease.

As used herein, specific activity refers to Units of activity per mgprotein. The milligrams of hyaluronidase is defined by the absorption ofa solution of at 280 nm assuming a molar extinction coefficient ofapproximately 1.7, in units of M⁻¹ cm⁻¹.

As used herein, elevated hyaluronan levels refers to amounts ofhyaluronan in a particular tissue, body fluid or cell, dependent uponthe disease or condition. For example, as a consequence of the presenceof a hyaluronan-rich tumor, hyaluronan (HA) levels can be elevated inbody fluids, such as blood, urine, saliva and serum, and/or in thetumorous tissue or cell. The level can be compared to a standard orother suitable control, such as a comparable sample from a subject whodoes not have the HA-associated disease.

As used herein, “activity” refers to a functional activity or activitiesof a polypeptide or portion thereof associated with a full-length(complete) protein. For example, active fragments of a polypeptide canexhibit an activity of a full-length protein. Functional activitiesinclude, but are not limited to, biological activity, catalytic orenzymatic activity, antigenicity (ability to bind or compete with apolypeptide for binding to an anti-polypeptide antibody),immunogenicity, ability to form multimers, and the ability tospecifically bind to a receptor or ligand for the polypeptide.

As used herein, “hyaluronidase activity” refers to the ability toenzymatically catalyze the cleavage of hyaluronic acid. The UnitedStates Pharmacopeia (USP) XXII assay for hyaluronidase determineshyaluronidase activity indirectly by measuring the amount of highermolecular weight hyaluronic acid, or hyaluronan, (HA) substrateremaining after the enzyme is allowed to react with the HA for 30 min at37° C. (USP XXII-NF XVII (1990) 644-645 United States PharmacopeiaConvention, Inc, Rockville, Md.). A Reference Standard solution can beused in an assay to ascertain the relative activity, in units, of anyhyaluronidase. In vitro assays to determine the hyaluronidase activityof hyaluronidases, such as PH20, including soluble PH20 and esPH20, areknown in the art and described herein. Exemplary assays include themicroturbidity assay that measures cleavage of hyaluronic acid byhyaluronidase indirectly by detecting the insoluble precipitate formedwhen the uncleaved hyaluronic acid binds with serum albumin and thebiotinylated-hyaluronic acid assay that measures the cleavage ofhyaluronic acid indirectly by detecting the remainingbiotinylated-hyaluronic acid non-covalently bound to microtiter platewells with a streptavidin-horseradish peroxidase conjugate and achromogenic substrate. Reference Standards can be used, for example, togenerate a standard curve to determine the activity in Units of thehyaluronidase being tested.

As used herein, “neutral active” refers to the ability of a PH20polypeptide to enzymatically catalyze the cleavage of hyaluronic acid atneutral pH (e.g. at or about pH 7.0). Generally, a neutral active andsoluble PH20, e.g., C-terminally truncated or N-partially glycosylatedPH20, has or has about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%,300%, 400%, 500%, 1000% or more activity compared to the hyaluronidaseactivity of a corresponding neutral active PH20 that is not C-terminallytruncated or N-partially glycosylated.

As used herein, a “GPI-anchor attachment signal sequence” is aC-terminal sequence of amino acids that directs addition of a preformedGPI-anchor to the polypeptide within the lumen of the ER. GPI-anchorattachment signal sequences are present in the precursor polypeptides ofGPI-anchored polypeptides, such as GPI-anchored PH20 polypeptides. TheC-terminal GPI-anchor attachment signal sequence typically contains apredominantly hydrophobic region of 8-20 amino acids, preceded by ahydrophilic spacer region of 8-12 amino acids, immediately downstream ofthe ω-site, or site of GPI-anchor attachment. GPI-anchor attachmentsignal sequences can be identified using methods well known in the art.These include, but are not limited to, in silico methods and algorithms(see, e.g. Udenfriend et al. (1995) Methods Enzymol. 250:571-582,Eisenhaber et al., (1999) J. Biol. Chem. 292: 741-758, Fankhauser etal., (2005) Bioinformatics 21:1846-1852, Omaetxebarria et al., (2007)Proteomics 7:1951-1960, Pierleoni et al., (2008) BMC Bioinformatics9:392), including those that are readily available on bioinformaticwebsites, such as the ExPASy Proteomics tools site (e.g., theWorldWideWeb site expasy.ch/tools/).

As used herein, “nucleic acids” include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is greater thanor equal to 2 amino acids in length, and less than or equal to 40 aminoacids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 1). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH2refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted 37 C.F.R. §§1.821-1.822, abbreviations for amino acid residuesare shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

All amino acid residue sequences represented herein by formulae have aleft to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is defined to include the amino acids listed in the Table ofCorrespondence (Table 1) and modified and unusual amino acids, such asthose referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein byreference. Furthermore, it should be noted that a dash at the beginningor end of an amino acid residue sequence indicates a peptide bond to afurther sequence of one or more amino acid residues, to anamino-terminal group such as NH₂ or to a carboxyl-terminal group such asCOOH.

As used herein, the “naturally occurring α-amino acids” are the residuesof those 20α-amino acids found in nature which are incorporated intoprotein by the specific recognition of the charged tRNA molecule withits cognate mRNA codon in humans. Non-naturally occurring amino acidsthus include, for example, amino acids or analogs of amino acids otherthan the 20 naturally-occurring amino acids and include, but are notlimited to, the D-isostereomers of amino acids. Exemplary non-naturalamino acids are described herein and are known to those of skill in theart.

As used herein, a DNA construct is a single- or double-stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g. Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exists a number of methodsto measure identity between two polynucleotide or polypeptides, the term“identity” is well known to skilled artisans (Carrillo, H. & Lipton, D.,SIAM J Applied Math 48:1073 (1988)).

As used herein, homologous (with respect to nucleic acid and/or aminoacid sequences) means about greater than or equal to 25% sequencehomology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%,80%, 85%, 90% or 95% sequence homology; the precise percentage can bespecified if necessary. For purposes herein the terms “homology” and“identity” are often used interchangeably, unless otherwise indicated.In general, for determination of the percentage homology or identity,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carrillo et al. (1988) SIAM J Applied Math 48:1073). By sequencehomology, the number of conserved amino acids is determined by standardalignment algorithms programs, and can be used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules would hybridize typically at moderate stringency or athigh stringency all along the length of the nucleic acid of interest.Also contemplated are nucleic acid molecules that contain degeneratecodons in place of codons in the hybridizing nucleic acid molecule.

Whether any two molecules have nucleotide sequences or amino acidsequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% “identical” or “homologous” can be determined using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA (Altschul, S. F., et al., J Mol Biol 215:403 (1990)); Guide toHuge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carrillo et al. (1988) SIAM J Applied Math 48:1073). For example,the BLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.). Percent homology or identity ofproteins and/or nucleic acid molecules can be determined, for example,by comparing sequence information using a GAP computer program (e.g.,Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith andWaterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP programdefines similarity as the number of aligned symbols (i.e., nucleotidesor amino acids), which are similar, divided by the total number ofsymbols in the shorter of the two sequences. Default parameters for theGAP program can include: (1) a unary comparison matrix (containing avalue of 1 for identities and 0 for non-identities) and the weightedcomparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, asdescribed by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE ANDSTRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979);(2) a penalty of 3.0 for each gap and an additional 0.10 penalty foreach symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” or “homology” representsa comparison between a test and a reference polypeptide orpolynucleotide. As used herein, the term at least “90% identical to”refers to percent identities from 90 to 99.99 relative to the referencenucleic acid or amino acid sequence of the polypeptide. Identity at alevel of 90% or more is indicative of the fact that, assuming forexemplification purposes a test and reference polypeptide length of 100amino acids are compared. No more than 10% (i.e., 10 out of 100) of theamino acids in the test polypeptide differs from that of the referencepolypeptide. Similar comparisons can be made between test and referencepolynucleotides. Such differences can be represented as point mutationsrandomly distributed over the entire length of a polypeptide or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g. 10/100 amino acid difference (approximately 90%identity). Differences are defined as nucleic acid or amino acidsubstitutions, insertions or deletions. At the level of homologies oridentities above about 85-90%, the result should be independent of theprogram and gap parameters set; such high levels of identity can beassessed readily, often by manual alignment without relying on software.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated thatcertain nucleic acid molecules can serve as a “probe” and as a “primer.”A primer, however, has a 3′ hydroxyl group for extension. A primer canbe used in a variety of methods, including, for example, polymerasechain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR,multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the 5′ end of a sequence to beamplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizeswith the complement of the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application. Complementary, whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, typically with less than 25%,15% or 5% mismatches between opposed nucleotides. If necessary, thepercentage of complementarity will be specified. Typically the twomolecules are selected such that they will hybridize under conditions ofhigh stringency.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wildtype form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least 80%, 90% or greater amino acid identity with a wildtypeand/or predominant form from the same species; the degree of identitydepends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90% or 95% identity or greater with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide. Reference to anallelic variant herein generally refers to variations in proteins amongmembers of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude modifications such as substitutions, deletions and insertions ofnucleotides. An allele of a gene also can be a form of a gene containinga mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human. For example for PH20, exemplary of species variants providedherein are primate PH20, such as, but not limited to, human, chimpanzee,macaque and cynomologous monkey. Generally, species variants have 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or sequenceidentity. Corresponding residues between and among species variants canbe determined by comparing and aligning sequences to maximize the numberof matching nucleotides or residues, for example, such that identitybetween the sequences is equal to or greater than 95%, equal to orgreater than 96%, equal to or greater than 97%, equal to or greater than98% or equal to greater than 99%. The position of interest is then giventhe number assigned in the reference nucleic acid molecule. Alignmentcan be effected manually or by eye, particularly, where sequenceidentity is greater than 80%.

As used herein, a human protein is one encoded by a nucleic acidmolecule, such as DNA, present in the genome of a human, including allallelic variants and conservative variations thereof. A variant ormodification of a protein is a human protein if the modification isbased on the wildtype or prominent sequence of a human protein.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements (e.g. substitutions) of amino acids and nucleotides,respectively. Exemplary of modifications are amino acid substitutions.An amino-acid substituted polypeptide can exhibit 65%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity toa polypeptide not containing the amino acid substitutions. Amino acidsubstitutions can be conservative or non-conservative. Generally, anymodification to a polypeptide retains an activity of the polypeptide.Methods of modifying a polypeptide are routine to those of skill in theart, such as by using recombinant DNA methodologies.

As used herein, suitable conservative substitutions of amino acids areknown to those of skill in the art and can be made generally withoutaltering the biological activity of the resulting molecule. Those ofskill in the art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224). Such substitutions can be made in accordance withthose set forth in TABLE 2 as follows:

TABLE 2 Original residue Exemplary conservative substitution Ala (A)Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; ValLys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; LeuOther substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

Hence, reference to a substantially purified polypeptide, such as asubstantially purified soluble PH20. refers to preparations of proteinsthat are substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of enzyme proteins having less that about 30% (by dryweight) of non-enzyme proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-enzymeproteins or 10% of non-enzyme proteins or less that about 5% ofnon-enzyme proteins. When the enzyme protein is recombinantly produced,it also is substantially free of culture medium, i.e., culture mediumrepresents less than about or at 20%, 10% or 5% of the volume of theenzyme protein preparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of enzyme proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of enzyme proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-enzymechemicals or components.

As used herein, synthetic, with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, production by recombinant means or using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce a heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal. Expression vectors are generally derived fromplasmid or viral DNA, or can contain elements of both. Thus, anexpression vector refers to a recombinant DNA or RNA construct, such asa plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein, “operably” or “operatively linked” when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiatesdownstream of the promoter and upstream of any transcribed sequences.The promoter is usually the domain to which the transcriptionalmachinery binds to initiate transcription and proceeds through thecoding segment to the terminator.

As used herein the term “assessing” is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protein, such as an enzyme, or a domainthereof, present in the sample, and also of obtaining an index, ratio,percentage, visual or other value indicative of the level of theactivity. Assessment can be direct or indirect. For example, thechemical species actually detected need not of course be theenzymatically cleaved product itself but can for example be a derivativethereof or some further substance. For example, detection of a cleavageproduct can be a detectable moiety such as a fluorescent moiety.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of ahyaluronidase enzyme is its degradation of hyaluronic acid.

As used herein equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions that do not substantially alter the activity orfunction of the protein or peptide. When equivalent refers to aproperty, the property does not need to be present to the same extent(e.g., two peptides can exhibit different rates of the same type ofenzymatic activity), but the activities are usually substantially thesame.

As used herein, “modulate” and “modulation” or “alter” refer to a changeof an activity of a molecule, such as a protein. Exemplary activitiesinclude, but are not limited to, biological activities, such as signaltransduction. Modulation can include an increase in the activity (i.e.,up-regulation or agonist activity), a decrease in activity (i.e.,down-regulation or inhibition) or any other alteration in an activity(such as a change in periodicity, frequency, duration, kinetics or otherparameter). Modulation can be context dependent and typically modulationis compared to a designated state, for example, the wildtype protein,the protein in a constitutive state, or the protein as expressed in adesignated cell type or condition.

As used herein, a composition refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms. Diseases and disorders ofinterest herein are hyaluronan-associated diseases and disorders.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease.

As used herein, a pharmaceutically effective agent, includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, chemotherapeutics, anesthetics, vasoconstrictors,dispersing agents, conventional therapeutic drugs, including smallmolecule drugs and therapeutic proteins.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, therapeutic effect means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition. A therapeutically effective amount refers to the amount of acomposition, molecule or compound which results in a therapeutic effectfollowing administration to a subject.

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a patient refers to a human subject exhibiting symptomsof a disease or disorder.

As used herein, about the same means within an amount that one of skillin the art would consider to be the same or to be within an acceptablerange of error. For example, typically, for pharmaceutical compositions,within at least 1%, 2%, 3%, 4%, 5% or 10% is considered about the same.Such amount can vary depending upon the tolerance for variation in theparticular composition by subjects.

As used herein, dosing regime refers to the amount of agent, forexample, the composition containing an anti-hyaluronan agent, forexample a soluble hyaluronidase or other agent, administered, and thefrequency of administration. The dosing regime is a function of thedisease or condition to be treated, and thus can vary.

As used herein, frequency of administration refers to the time betweensuccessive administrations of treatment. For example, frequency can bedays, weeks or months. For example, frequency can be more than onceweekly, for example, twice a week, three times a week, four times aweek, five times a week, six times a week or daily. Frequency also canbe one, two, three or four weeks. The particular frequency is functionof the particular disease or condition treated. Generally, frequency ismore than once weekly, and generally is twice weekly.

As used herein, a “cycle of administration” refers to the repeatedschedule of the dosing regime of administration of the enzyme and/or asecond agent that is repeated over successive administrations. Forexample, an exemplary cycle of administration is a 28 day cycle withadministration twice weekly for three weeks, followed by one-week ofdiscontinued dosing.

As used herein, when referencing dosage based on mg/kg of the subject,an average human subject is considered to have a mass of about 70 kg-75kg, such as 70 kg.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms or,adverse effects of a condition, such as, for example, reduction ofadverse effects associated with or that occur upon administration of ananti-hyaluronan agent, such as a PEGylated hyaluronidase.

As used herein, prevention or prophylaxis refers to a reduction in therisk of developing a disease or condition.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect. Hence, it is thequantity necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, unit dose form refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, a single dosage formulation refers to a formulation as asingle dose.

As used herein, formulation for direct administration means that thecomposition does not require further dilution for administration.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass anti-hyaluronan agents, for example hyaluronan degradingenzyme, such as hyaluronidase, and second agent compositions containedin articles of packaging. For example, a second agent is acorticosteroid.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a combination, such as a combination of compositionsprovided herein, refers to an association of elements of thecombination.

As used herein a kit refers to a combination of components, such as acombination of the compositions herein and another item for a purposeincluding, but not limited to, reconstitution, activation, andinstruments/devices for delivery, administration, diagnosis, andassessment of a biological activity or property. Kits optionally includeinstructions for use.

As used herein, a cellular extract or lysate refers to a preparation orfraction which is made from a lysed or disrupted cell.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; pigs and other animals. Non-human animals exclude humans asthe contemplated animal. The hyaluronidases provided herein are from anysource, animal, plant, prokaryotic and fungal. Most hyaluronidases areof animal origin, including mammalian origin. Generally hyaluronidasesare of human origin.

As used herein, anti-cancer treatments include administration of drugsand other agents for treating cancer, and also treatment protocols, suchas surgery and radiation. Anti-cancer treatments include administrationof anti-cancer agents.

As used herein, an anti-cancer agent refers to any agents, or compounds,used in anti-cancer treatment. These include any agents, when used aloneor in combination with other compounds, that can alleviate, reduce,ameliorate, prevent, or place or maintain in a state of remission ofclinical symptoms or diagnostic markers associated with tumors andcancer, and can be used in combinations and compositions providedherein. Exemplary anti-cancer agents include, but are not limited to,anti-hyaluronan agents, such as the PEGylated hyaluronan degradingenzymes provided herein used singly or in combination and/or incombination with other anti-cancer agents, such as chemotherapeutics,polypeptides, antibodies, peptides, small molecules or gene therapyvectors, viruses or DNA.

As used herein, a control refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a compound comprising or containing “anextracellular domain” includes compounds with one or a plurality ofextracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. Overview

1. Anti-Hyaluronan Agents and Hyaluronan-Associated Diseases, Conditionsand/or Disorders

Certain diseases are associated with expression and/or production ofhyaluronan, including inflammatory diseases and cancers. HA is linked toa variety of biological processes involved with progression of suchdiseases (see e.g. Itano et al. (2008) Semin Cancer Biol 18(4):268-274;Tammi et al. (2008) Semin Cancer Biol 18(4):288-295). For example, HA islinked to biological processes associated with tumor progression,including epithelial-mesenchymal transition, and the p53 tumorsuppressor pathway. Also, HA is involved in increased water uptake andinterstitial fluid pressure (IFP) in disease tissues, such as tumors,thereby resulting in compressed tumor vasculature. For example, at sitesof inflammation or in a tumor focus, there is rapid accumulation ofhyaluronan, other matrix components and water. Because of this rapidaccumulation, the diseased site cannot come to equilibrium with itsenvironment and therefore has a higher interstitial fluid pressure thannormal tissues.

Anti-hyaluronan agents reduce hyaluronic acid (HA; also referred toherein as hyaluronan) levels by interfering with its synthesis orincreasing its degradation. For example, hyaluronan degrading enzymes,such as hyaluronidase, interfere with and degrade hyaluronic acid (HA).Treatment with agents that degrade or inhibit hyaluronan synthesis, suchas hyaluronan degrading enzymes, reduce the hyaluronan such that thetissue deflates, the blood vessels expand, and more blood can flowthrough the site.

In addition, the IFP of most solid tumors and other diseased tissuesassociated with accumulated HA is elevated, acting as a barrier toefficient drug delivery (Heldin et al. (2004) Nat Rev Cancer4(10):806-813). Thus, in addition to diminishing IFP and water contentat the tissue site and associated increased vascular perfusion,anti-hyaluronan agents, such as hyaluronan degrading enzymes, also canenhance the activity of coadministered therapies. For example,anti-hyaluronan agents, such as hyaluronan degrading enzymes, canenhance the delivery of chemotherapeutic agents to tumors.

Hence, anti-hyaluronan agents, such as hyaluronan degrading enzymes,exhibit properties useful for single-agent or combination therapy ofdiseases and conditions that exhibit the accumulation of hyaluronan(hyaluronic acid, HA). Hyaluronan-associated diseases, conditions and/ordisorders, include, cancers and inflammatory diseases. Hyaluronan-richcancers include, but are not limited to, tumors, including solid tumors,for example, late-stage cancers, a metastatic cancers, undifferentiatedcancers, ovarian cancer, in situ carcinoma (ISC), squamous cellcarcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lungcancer, breast cancer, colon cancer and other cancers. Hyaluronidase hasbeen shown to remove HA from tumors resulting in the reduction of tumorvolume, the reduction of intratumoral interstitial pressure, the slowingof tumor cell proliferation, and the enhanced efficacy ofco-administered chemotherapeutic drugs and biological agents by enablingincreased tumor penetration (see e.g. U.S. published application No.20100003238 and International published PCT Appl. No WO 2009/128917).

PEGylation is an established technology used to increase the half-lifeof therapeutic proteins in the body thus enabling their use in systemictreatment protocols. PEGylation of anti-hyaluronan agents, such ashyaluronan degrading enzymes, such as hyaluronidase extends itshalf-life in the body from less than a minute to approximately 48 to 72hours and allows for the systemic treatment of tumors rich in HA (seee.g. U.S. published application No. 20100003238 and Internationalpublished PCT Appl. No WO 2009/128917). The increased half-life relativeto unPEGylated hyaluronidase, permits not only removal of HA, but also,due to its continued presence in the plasma and its ability to degradeHA, reduces or decreases the extent of regeneration of HA withindiseased tissues, such as the tumor. Maintenance of plasma enzyme levelscan remove HA, such as tumor HA, and also counteract HA resynthesis.

2. Adverse Effects Associated with Treatment with Anti-Hyaluronan Agents

The use of anti-hyaluronan agents, for example hyaluronan degradingenzymes, such as hyaluronidase, has not previously been observed to beassociated with side effects. Nevertheless, as described herein, severalspecies, including humans, developed musculoskeletal side effects afterbeing dosed with PEGylated hyaluronidase, as observed in bothpreclinical and clinical studies (described herein below and in Examples6-7). The musculoskeletal side effects included stiffness, muscle andjoint pain, and a decrease in range of motion at knee and elbow joints.

Humans who received a single intravenous dose of 0.05 mg/kg PEGPH20manifested adverse musculoskeletal symptoms approximately six hoursafter dosing. The adverse effects included one or more of the followingeffects: muscle and joint pain/stiffness of upper and lower extremities,cramping, muscle, myositis muscle soreness and tenderness over theentire body, weakness and fatigue. On the Common Terminology Criteriafor Adverse Events (CTCAE) scale, symptoms were observed to reach Grade3. The adverse effects resolved over several days once treatment wasdiscontinued. Example 6 further exemplifies adverse events occurring ina sample of human patients treated with PEGPH20 under various dosageregimens, dose and dosing frequency. The severity of the musculoskeletalevents appear to be influenced by the combination of PEGPH20 dose anddosing frequency. For example, the symptoms appear to be more severewith higher doses of PEGPH20 and a more frequent dosing schedule.

Monkeys who were administered PEGPH20 exhibited hypoactivity, lethargy,disorientation, hunched posture and a decrease in limb joint angle. Fourweeks of a twice-weekly IV dosing with PEGPH20 resulted in a decrease inthe range of motion at the knee and elbow joints. After dosing wasdiscontinued, partial to full recovery was observed.

Beagle dogs that received a single dose of either 0.08 mg/kg PEGPH20 or0.3 mg/kg PEGPH20 exhibited on the following day reduced walkingability, difficulty standing, decreased activity and tightness of themuscles of the neck, back and extremities. The animals completelyrecovered by day four after dosing was discontinued.

3. Use of Corticosteroids to Ameliorate the Adverse Effects ofAnti-Hyaluronan Agents

Provided herein are methods and uses to ameliorate or prevent adverseeffects, such as musculoskeletal side effects, associated with treatmentor administration with an anti-hyaluronan agent by premedication orco-medication with a corticosteroid. For example, as shown herein, themusculoskeletal side effects observed with systemic administration ofPEGylated hyaluronidase can be ameliorated, and/or eliminated, bypremedication and/or co-medication with a corticosteroid, e.g., aglucocorticoid such as dexamethasone. The result is an improvedtolerability of higher PEGPH20 doses and dosing frequency combinations.Hence, as described herein, adverse events associated with singletherapy or combination therapy PH20 treatment to treat ahyaluronan-associated disease or condition can be achieved in thepresence of a corticosteroid.

Hence, provided herein are methods to ameliorate adverse effectsassociated with the use of an anti-hyaluronan agent, such as a PEGylatedhyaluronan degrading enzyme, for single-agent or combination therapy ofdiseases and conditions which exhibit the accumulation of hyaluronan.The methods use corticosteroids, e.g., glucocorticoids, to amelioratethe adverse musculoskeletal side effects of treatment withanti-hyaluronan agents, such as PEGylated hyaluronan degrading enzymes.In some examples, the adverse side effects are reduced. In otherexamples, the adverse side effects are eliminated. Typically, thecorticosteroid is administered orally, although any method ofadministration of the corticosteroid is contemplated. Typically, theglucocorticoid is administered at an amount between at or about 0.4 and20 mgs, for example, at or about 0.4 mgs, 0.5 mgs, 0.6 mgs, 0.7 mgs,0.75 mgs, 0.8 mgs, 0.9 mgs, 1 mg, 2 mgs, 3 mgs, 4 mgs, 5 mgs, 6 mgs, 7mgs, 8 mgs, 9 mgs, 10 mgs, 11 mgs, 12 mgs, 13 mgs, 14 mgs, 15 mgs, 16mgs, 17 mgs, 18 mgs, 19 mgs or 20 mgs per dose.

The corticosteroid can be administered before, after or concurrentlywith the anti-hyaluronan agents, such as a PEGylated hyaluronidase. Insome examples, the corticosteroid is administered prior to theadministration of the anti-hyaluronan agent, such as a PEGylatedhyaluronan degrading enzyme. For example, the corticosteroid can beadministered up to at or about 0.5 minutes, 1 minute, 2 minutes, 3minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36hours or more prior to administration of the anti-hyaluronan agent, forexample, a PEGylated hyaluronan degrading enzyme. In other examples, thecorticosteroid is administered at the same time as administration of theanti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme. In this example, the corticosteroid can be administeredseparately or together with the anti-hyaluronan agent. Typically, thecorticosteroid is administered separately from the anti-hyaluronanagent. In other examples, the corticosteroid is administered subsequentto the administration of the anti-hyaluronan agent, for example aPEGylated hyaluronan degrading enzyme. For example, the corticosteroidcan be administered up to at or about 0.5 minutes, 1 minute, 2 minutes,3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36hours or more after the administration of the anti-hyaluronan agent,such as a PEGylated hyaluronan degrading enzyme.

In some examples, the corticosteroid is used in a dosing regimen wherethe anti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme, is administered alone for treatment of a hyaluronan associateddisease or condition. In other examples, the corticosteroid is used in adosing regiment where the anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzymes, is administered in combination with one ormore additional agents and/or treatment for treating the disease ordisorder, for example, an anti-cancer agent, such as a chemotherapy,antibody, vector or nucleic acid for treating cancer. In this example,the second treatment or agent can be administered separately or togetherwith the anti-hyaluronan agent. For example, the anti-hyaluronan agent,for example a PEGylated hyaluronan degrading enzyme or other modifiedhyaluonan-degrading enzyme, are administered before, after or with anadditional agent or treatment. Hence, anti-hyaluronan agents, forexample hyaluronan degrading enzymes, particularly modified hyaluronandegrading enzymes, such as PEGylated soluble hyaluronidases, can beadministrated as therapeutic agents alone or in combination with othertherapeutic agents.

C. Anti-Hyaluronan Agents

HA, also called hyaluronic acid, hyaluronate or hyaluronan, is a highmolecular weight linear glycosaminoglycan that contains repeatingdisaccharide units, β1,3 N-acetyl-D-glucosamine-linked β1,4 toD-glucoronic acid. Hyaluronan is widely distributed throughoutconnective, epithelial, and neural tissues. It also is a major componentof the extracellular matrix and a constituent of the interstitialbarrier. For example, the extracellular matrix of cartilage contains asmall amount of HA. HA plays a role in tissue remodeling duringdevelopment and normal tissue homeostatis, and likely is involved incell adhesion and migration. HA also functions as a biological lubricantin joints and is important both during movement and under staticconditions (see e.g. Engstrom-Laurent (1997) J. Intern. Med., 242:57-60;Jiang et al. (2007) Ann. Rev. Cell Dev. Biol., 23:435-61). HA can beused itself to modulate disease, for example, in the treatment of jointdisease, ophthalmic surgical device or in wound healing.

HA also is involved in disease. HA accumulation, such as by alteredhyaluronan metabolism, distribution and function is associated witharthritis, immune and inflammatory disorders, pulmonary and vasculardiseases and cancer (Morohashi et al. (2006) Biochem. Biophys. Res.Comm., 345:1454-1459). Such diseases can be treated by inhibiting HAsynthesis or degrading HA (see e.g. Morohashi 2006; U.S. publishedapplication No. 20100003238 and International published PCT Appl. No WO2009/128917). For example, anti-hyaluronan agents, such as hyaluronandegrading enzymes, can be used to treat hyaluronan associated diseasesor conditions, including tumors and cancers or inflammatory diseases orconditions. As disclosed herein, such treatments that reduce hyaluronanlevels on cells and tissues can be associated with adverse side effects,such as musculoskeletal side effects. As provided herein, these adverseeffects can be alleviated or ameliorated by pre-treatment orco-treatment with corticosteroids.

Hence, corticosteroids can be used in methods to reduce adverse sideeffects, such as musculoskeletal side effects, of any agent thatinhibits, degrades or reduces HA accumulation or elevation in diseasestates. Such agents are known to one of skill in the art or can beidentified. For example, effects of agents, including effects of doseand route of administration, on HA synthesis or degradation can beassessed in various assays known to one of skill in the art, includingbut not limited to any described herein or known in the art, forexample, in vitro assays that measure hyaluronan degradation (see e.g.Frost and Stern (1997) Anal. Biochem. 251:263-269), staining tissue orother samples for HA such as by using an HA-binding protein or otheranti-HA reagent (see e.g. Nishida et al. (1999) J. Biol. Chem.,274:21893-21899), particle exclusion assay (Nishida et al. 1999;Morohashi et al. (2006) Biochem Biophys. Res. Comm., 345:1454-1459);measuring or assessing HAS mRNA expression for an has gene (Nishida etal. 1999). Exemplary of such anti-hyaluronan agents are agents thatinhibit hyaluronan synthesis or degrade hyaluronan.

1. Agents that Inhibit Hyaluronan Synthesis

HA can be synthesized by three enzymes that are the products of threerelated mammalian genes identified as HA synthases, designated has-1,has-2 and has-3. Different cell types express different HAS enzymes andexpression of HAS mRNAs is correlated with HA biosynthesis. It is knownthat silencing HAS genes in tumor cells inhibits tumor growth andmetastasis. An anti-hyaluronan agent includes any agent that inhibits,reduces or downregulates the expression or level of an HA synthase. Suchagents are known to one of skill in the art or can be identified.

For example, downregulation of a HAS can be accomplished by providingoligonucleotides that specifically hybridize or otherwise interact withone or more nucleic acid molecules encoding an HAS. For example,anti-hyaluronan agents that inhibit hyaluronan synthesis includeantisense or sense molecules against an has gene. Such antisense orsense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded or otherwise renderedinoperable. In other examples, post-transcriptional gene silencing(PTGS), RNAi, ribozymes and DNAzymes can be employed. It is within thelevel of one skill in the art to generate such constructs based on thesequence of HAS1 (set forth in SEQ ID NO:195), HAS2 (set forth in SEQ IDNO:196) or HAS3 (set forth in SEQ ID NO:197 or 198). It is understood inthe art that the sequence of an antisense or sense compound need not be100% complementary to that of its target nucleic acid to be specificallyhybridizable. Moreover, an oligonucleotide may hybridize over one ormore segments such that intervening or adjacent segments are notinvolved in the hybridization event (e.g. a loop structure or hairpinstructure). Generally, the antisense or sense compounds have at least70% sequence complementarity to a target region within the targetnucleic acid, for example, 75% to 100% complementarity, such as 75%,80%, 85%, 90%, 95% or 100%. Exemplary sense or antisense molecules areknown in the art (see e.g. Chao et al. (2005) J. Biol. Chem.,280:27513-27522; Simpson et al. (2002) J. Biol. Chem., 277:10050-10057;Simpson et al. (2002) Am. J Path., 161:849; Nishida et al. (1999) J.Biol. Chem., 274:21893-21899; Edward et al. (2010) British JDermatology, 162:1224-1232; Udabage et al. (2005) Cancer Res., 65:6139;and published U.S. Patent application No. US20070286856).

Another exemplary anti-hyaluronan agent that is an HA synthesisinhibitor is 4-Methylumbelliferone (4-MU; 7-hydroxy-4-methylcoumarin) ora derivative thereof. 4-MU acts by reducing the UDP-GlcUA precursor poolthat is required for HA synthesis. For example, in mammalian cells, HAis synthesized by HAS using UDP-glucuronic acid (UGA) andUDP-N-acetyl-D-glucosamine precursors. 4-MU interferes with the processby which UGA is generated, thereby depleting the intracellular pool ofUGA and resulting in inhibition of HA synthesis. 4-MU is known to haveantitumor activity (see e.g. Lokeshwar et al. (2010) Cancer Res.,70:2613-23; Nakazawa et al. (2006) Cancer Chemother. Pharmacol.,57:165-170; Morohashi et al. (2006) Biochem. Biophys. Res. Comm.,345-1454-1459). Oral administration of 4-MU at 600 mg/kg/d) reducesmetastases by 64% in the B16 melanoma model (Yoshihara et al. (2005)FEBS Lett., 579:2722-6). The structure of 4-MU is set forth below. Also,derivatives of 4-MU exhibit anti-cancer activity, in particular6,7-dihydrozy-4-methyl coumarin and 5,7-dihydroxy-4-methyl coumarin (seee.g. Morohashi et al. (2006) Biochem. Biophys. Res. Comm.,345-1454-1459).

4-Methylumbelliferone (4-MU; C₁₀H₈O₃)

Further exemplary anti-hyaluronan agents are tyrosine kinase inhibitors,such as Leflunomide (Arava), genistein or erbstatin. Leflunomide also isa pyrimidine synthesis inhibitor. Leflunomide is a known drug for thetreatment of Rheumatoid arthritis (RA), and also is effective intreating the rejection of allografts as well as xenografts. HA is knownto directly or indirectly contribute to HA (see e.g. Stuhlmeier (2005) JImmunol., 174:7376-7382). Tyrosine kinase inhibitors inhibit HAS1 geneexpression (Stuhlmeier 2005).

2. Hyaluronan Degrading Enzymes

Anti-hyaluronan agents include hyaluronan degrading enzymes. Hyaluronanis an essential component of the extracellular matrix and a majorconstituent of the interstitial barrier. By catalyzing the hydrolysis ofhyaluronan, hyaluronan degrading enzymes lower the viscosity ofhyaluronan, thereby increasing tissue permeability and increasing theabsorption rate of fluids administered parenterally. As such, hyaluronandegrading enzymes, such as hyaluronidases, have been used, for example,as spreading or dispersing agents in conjunction with other agents,drugs and proteins to enhance their dispersion and delivery.

Hyaluronan degrading enzymes act to degrade hyaluronan by cleavinghyaluronan polymers, which are composed of repeating disaccharidesunits, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc),linked together via alternating β-1→4 and β-1→3 glycosidic bonds.Hyaluronan chains can reach about 25,000 disaccharide repeats or more inlength and polymers of hyaluronan can range in size from about 5,000 to20,000,000 Da in vivo. Accordingly, hyaluronan degrading enzymes for theuses and methods provided include any enzyme having the ability tocatalyze the cleavage of a hyaluronan disaccharide chain or polymer. Insome examples the hyaluronan degrading enzyme cleaves the β-1→4glycosidic bond in the hyaluronan chain or polymer. In other examples,the hyaluronan degrading enzyme catalyze the cleavage of the β-1→3glycosidic bond in the hyaluronan chain or polymer.

Hence, hyaluronan degrading enzymes, such as hyaluronidases, are afamily of enzymes that degrade hyaluronic acid, which is an essentialcomponent of the extracellular matrix and a major constituent of theinterstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, amajor constituent of the interstitial barrier, hyaluronan degradingenzymes lower the viscosity of hyaluronic acid, thereby increasingtissue permeability. As such, hyaluronan degrading enzymes, such ashyaluronidases, have been used, for example, as a spreading ordispersing agent in conjunction with other agents, drugs and proteins toenhance their dispersion and delivery. Hyaluronan-degrading enzymes alsoare used as an adjuvant to increase the absorption and dispersion ofother injected drugs, for hypodermoclysis (subcutaneous fluidadministration), and as an adjunct in subcutaneous urography forimproving resorption of radiopaque agents. Hyaluronan-degrading enzymes,for example, hyaluronidase can be used in applications of ophthalmicprocedures, for example, peribulbar and sub-Tenon's block in localanesthesia prior to ophthalmic surgery. Hyaluronidase also can be use inother therapeutic and cosmetic uses, for example, by promoting akinesiain cosmetic surgery, such as blepharoplasties and face lifts.

Various forms of hyaluronan degrading enzymes, including hyaluronidaseshave been prepared and approved for therapeutic use in subjects,including humans. The provided compositions and methods can be used, viathese and other therapeutic uses, to treat hyaluronan-associateddiseases and conditions. For example, animal-derived hyaluronidasepreparations include Vitrase® (ISTA Pharmaceuticals), a purified ovinetesticular hyaluronidase, Amphadase® (Amphastar Pharmaceuticals), abovine testicular hyaluronidase and Hydase™ (Prima Pharm Inc.), a bovinetesticular hyaluronidase. It is understood that any animal-derivedhyaluronidase preparation can be used in the methods and uses providedherein (see, e.g., U.S. Pat. Nos. 2,488,564, 2,488,565, 2,676,139,2,795,529, 2,806,815, 2,808,362, 5,747,027 and 5,827,721 and InternationPCT Application No. WO2005/118799). Hylenex® (Halozyme Therapeutics) isa human recombinant hyaluronidase produced by genetically engineeredChinese Hamster Ovary (CHO) cells containing nucleic acid encodingsoluble forms of PH20, designated rHuPH20.

Exemplary of hyaluronan degrading enzymes in the compositions andmethods provided herein are soluble hyaluronidases. Other exemplaryhyaluronan degrading enzymes include, but are not limited to particularchondroitinases and lyases that have the ability to cleave hyaluronan.

As described below, hyaluronan-degrading enzymes exist in membrane-boundor soluble forms that are secreted from cells. For purposes herein,soluble hyaluronan-degrading enzymes are provided for use in themethods, uses, compositions or combinations herein. Thus, wherehyaluronan-degrading enzymes include a glycosylphosphatidylinositol(GPI) anchor and/or are otherwise membrane-anchored or insoluble, suchhyaluronan-degrading enzymes are provided herein in soluble form bytruncation or deletion of the GPI anchor to render the enzyme secretedand soluble. Thus, hyaluronan-degrading enzymes include truncatedvariants, e.g. truncated to remove all or a portion of a GPI anchor.Hyaluronan-degrading enzymes provided herein also include allelic orspecies variants or other variants, of a soluble hyaluronan-degradingenzyme. For example, hyaluronan degrading enzymes can contain one ormore variations in its primary sequence, such as amino acidsubstitutions, additions and/or deletions. A variant of ahyaluronan-degrading enzyme generally exhibits at least or about 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity compared to the hyaluronan-degrading enzyme notcontaining the variation. Any variation can be included in thehyaluronan degrading enzyme for the purposes herein provided the enzymeretains hyaluronidase activity, such as at least or about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more of the activity of a hyaluronan degrading enzyme notcontaining the variation (as measured by in vitro and/or in vivo assayswell known in the art and described herein).

Where the methods and uses provided herein describe the use of a solublehyaluronidase, accordingly any hyaluronan degrading enzyme, generally asoluble hyaluronan degrading enzyme, can be used. It is understand thatany hyaluronidase can be used in the methods and uses provided herein(see, e.g., U.S. Pat. No. 7,767,429 and U.S. Publication Nos.US20040268425 and US20100143457).

a. Hyaluronidases

Hyaluronidases are members of a large family of hyaluronan degradingenzymes. There are three general classes of hyaluronidases:mammalian-type hyaluronidases, bacterial hyaluronidases andhyaluronidases from leeches, other parasites and crustaceans. Suchenzymes can be used in the compositions, combinations and methodsprovided herein.

i. Mammalian-Type Hyaluronidases

Mammalian-type hyaluronidases (EC 3.2.1.35) areendo-β-N-acetylhexosaminidases that hydrolyze the β-1→4 glycosidic bondof hyaluronan into various oligosaccharide lengths such astetrasaccharides and hexasaccharides. These enzymes have both hydrolyticand transglycosidase activities, and can degrade hyaluronan andchondroitin sulfates (CS), generally C4-S and C6-S. Hyaluronidases ofthis type include, but are not limited to, hyaluronidases from cows(bovine) (SEQ ID NOS:10, 11 and 64 and BH55 (U.S. Pat. Nos. 5,747,027and 5,827,721), nucleic acid molecules set forth in SEQ ID NOS:190-192),sheep (Ovis aries) (SEQ ID NO: 26, 27, 63 and 65, nucleic acid moleculesset forth in SEQ ID NOS:66 and 193-194), yellow jacket wasp (SEQ IDNOS:12 and 13), honey bee (SEQ ID NO:14), white-face hornet (SEQ IDNO:15), paper wasp (SEQ ID NO:16), mouse (SEQ ID NOS:17-19, 32), pig(SEQ ID NOS:20-21), rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25),orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ ID NO:29), guinea pig(SEQ ID NO:30), chimpanzee (SEQ ID NO:101), rhesus monkey (SEQ IDNO:102), and human hyaluronidases (SEQ. ID NOS:1-2, 36-39). Exemplary ofhyaluronidases in the compositions, combinations and methods providedherein are soluble hyaluronidases.

Mammalian hyaluronidases can be further subdivided into those that areneutral active, predominantly found in testes extracts, and acid active,predominantly found in organs such as the liver. Exemplary neutralactive hyaluronidases include PH20, including but not limited to, PH20derived from different species such as ovine (SEQ ID NOS:27, 63 and 65),bovine (SEQ ID NO:11 and 64) and human (SEQ ID NO:1). Human PH20 (alsoknown as SPAM1 or sperm surface protein PH20), is generally attached tothe plasma membrane via a glycosylphosphatidyl inositol (GPI) anchor. Itis naturally involved in sperm-egg adhesion and aids penetration bysperm of the layer of cumulus cells by digesting hyaluronic acid.

Besides human PH20 (also termed SPAM1), five hyaluronidase-like geneshave been identified in the human genome, HYAL1, HYAL2, HYAL3, HYAL4 andHYALP1. HYALP1 is a pseudogene, and HYAL3 (SEQ ID NO:38) has not beenshown to possess enzyme activity toward any known substrates. HYAL4(precursor polypeptide set forth in SEQ ID NO:39) is a chondroitinaseand exhibits little activity towards hyaluronan. HYAL1 (precursorpolypeptide set forth in SEQ ID NO:36) is the prototypical acid-activeenzyme and PH20 (precursor polypeptide set forth in SEQ ID NO:1) is theprototypical neutral-active enzyme. Acid-active hyaluronidases, such asHYAL1 and HYAL2 (precursor polypeptide set forth in SEQ ID NO:37)generally lack catalytic activity at neutral pH (i.e. pH 7). Forexample, HYAL1 has little catalytic activity in vitro over pH 4.5 (Frostet al. (1997) Anal. Biochem. 251:263-269). HYAL2 is an acid-activeenzyme with a very low specific activity in vitro. Thehyaluronidase-like enzymes also can be characterized by those which aregenerally attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor such as human HYAL2 and human PH20(Danilkovitch-Miagkova et al. (2003) Proc Natl Acad Sci USA100(8):4580-5), and those which are generally soluble such as humanHYAL1 (Frost et al. (1997) Biochem Biophys Res Commun. 236(1):10-5).

PH20

PH20, like other mammalian hyaluronidases, is anendo-β-N-acetyl-hexosaminidase that hydrolyzes the β1→4 glycosidic bondof hyaluronic acid into various oligosaccharide lengths such astetrasaccharides and hexasaccharides. It has both hydrolytic andtransglycosidase activities and can degrade hyaluronic acid andchondroitin sulfates, such as C4-S and C6-S. PH20 is naturally involvedin sperm-egg adhesion and aids penetration by sperm of the layer ofcumulus cells by digesting hyaluronic acid. PH20 is located on the spermsurface, and in the lysosome-derived acrosome, where it is bound to theinner acrosomal membrane. Plasma membrane PH20 has hyaluronidaseactivity only at neutral pH, while inner acrosomal membrane PH20 hasactivity at both neutral and acid pH. In addition to being ahyaluronidase, PH20 also appears to be a receptor for HA-induced cellsignaling, and a receptor for the zona pellucida surrounding the oocyte.

Exemplary PH20 proteins include, but are not limited to, human(precursor polypeptide set forth in SEQ ID NO:1, mature polypeptide setforth in SEQ ID NO: 2), chimpanzee (SEQ ID NO:101), Rhesus monkey (SEQID NO:102) bovine (SEQ ID NOS: 11 and 64), rabbit (SEQ ID NO: 25), ovinePH20 (SEQ ID NOS: 27, 63 and 65), Cynomolgus monkey (SEQ ID NO: 29),guinea pig (SEQ ID NO: 30), rat (SEQ ID NO: 31) and mouse (SEQ ID NO:32) PH20 polypeptides.

Bovine PH20 is a 553 amino acid precursor polypeptide (SEQ ID NO:11).Alignment of bovine PH20 with the human PH20 shows only weak homology,with multiple gaps existing from amino acid 470 through to therespective carboxy termini due to the absence of a GPI anchor in thebovine polypeptide (see e.g., Frost G I (2007) Expert Opin. Drug. Deliv.4: 427-440). In fact, clear GPI anchors are not predicted in many otherPH20 species besides humans. Thus, PH20 polypeptides produced from ovineand bovine naturally exist as soluble forms. Though bovine PH20 existsvery loosely attached to the plasma membrane, it is not anchored via aphospholipase sensitive anchor (Lalancette et al. (2001) Biol Reprod.65(2):628-36). This unique feature of bovine hyaluronidase has permittedthe use of the soluble bovine testes hyaluronidase enzyme as an extractfor clinical use (Wydase®, Hyalase®).

The human PH20 mRNA transcript is normally translated to generate a 509amino acid precursor polypeptide (SEQ ID NO:1) containing a 35 aminoacid signal sequence at the N-terminus (amino acid residue positions1-35) and a 19 amino acid glycosylphosphatidylinositol (GPI) anchorattachment signal sequence at the C-terminus (amino acid residuepositions 491-509). The mature PH20 therefore, is a 474 amino acidpolypeptide set forth in SEQ ID NO:2. Following transport of theprecursor polypeptide to the ER and removal of the signal peptide, theC-terminal GPI-attachment signal peptide is cleaved to facilitatecovalent attachment of a GPI anchor to the newly-formed C-terminal aminoacid at the amino acid position corresponding to position 490 of theprecursor polypeptide set forth in SEQ ID NO:1. Thus, a 474 amino acidGPI-anchored mature polypeptide with an amino acid sequence set forth inSEQ ID NO:2 is produced.

Human PH20 exhibits hyaluronidase activity at neutral and acid pH. Inone aspect, human PH20 is the prototypical neutral-active hyaluronidasethat is generally locked to the plasma membrane via a GPI anchor. Inanother aspect, PH20 is expressed on the inner acrosomal membrane whereit has hyaluronidase activity at neutral and acid pH. It appears thatPH20 contains two catalytic sites at distinct regions of thepolypeptide: the Peptide 1 and Peptide 3 regions (Cherr et aL, (2001)Matrix Biology 20:515-525). Evidence indicates that the Peptide 1 regionof PH20, which corresponds to amino acid positions 107-137 of the maturepolypeptide set forth in SEQ ID NO:2 and positions 142-172 of theprecursor polypeptide set forth in SEQ ID NO:1, is required for enzymeactivity at neutral pH. Amino acids at positions 111 and 113(corresponding to the mature PH20 polypeptide set forth in SEQ ID NO:2)within this region appear to be important for activity, as mutagenesisby amino acid replacement results in PH20 polypeptides with 3%hyaluronidase activity or undetectable hyaluronidase activity,respectively, compared to the wild-type PH20 (Arming et aL, (1997) Eur.J. Biochem. 247:810-814).

The Peptide 3 region, which corresponds to amino acid positions 242-262of the mature polypeptide set forth in SEQ ID NO:2, and positions277-297 of the precursor polypeptide set forth in SEQ ID NO: 1, appearsto be important for enzyme activity at acidic pH. Within this region,amino acids at positions 249 and 252 of the mature PH20 polypeptideappear to be essential for activity, and mutagenesis of either oneresults in a polypeptide essentially devoid of activity (Arming et al.,(1997) Eur. J. Biochem. 247:810-814).

In addition to the catalytic sites, PH20 also contains ahyaluronan-binding site. Experimental evidence indicate that this siteis located in the Peptide 2 region, which corresponds to amino acidpositions 205-235 of the precursor polypeptide set forth in SEQ ID NO: 1and positions 170-200 of the mature polypeptide set forth in SEQ IDNO:2. This region is highly conserved among hyaluronidases and issimilar to the heparin binding motif. Mutation of the arginine residueat position 176 (corresponding to the mature PH20 polypeptide set forthin SEQ ID NO:2) to a glycine results in a polypeptide with only about 1%of the hyaluronidase activity of the wild type polypeptide (Arming etal., (1997) Eur. J. Biochem. 247:810-814).

There are seven potential N-linked glycosylation sites in human PH20 atN82, N166, N235, N254, N368, N393, N490 of the polypeptide exemplifiedin SEQ ID NO: 1. Because amino acids 36 to 464 of SEQ ID NO:1 appear tocontain the minimally active human PH20 hyaluronidase domain, theN-linked glycosylation site N-490 is not required for properhyaluronidase activity. There are six disulfide bonds in human PH20. Twodisulfide bonds between the cysteine residues C60 and C351 and betweenC224 and C238 of the polypeptide exemplified in SEQ ID NO: 1(corresponding to residues C25 and C316, and C189 and C203 of the maturepolypeptide set forth in SEQ ID NO:2, respectively). A further fourdisulfide bonds are formed between between the cysteine residues C376and C387; between C381 and C435; between C437 and C443; and between C458and C464 of the polypeptide exemplified in SEQ ID NO: 1 (correspondingto residues C341 and C352; between C346 and C400; between C402 and C408;and between C423 and C429 of the mature polypeptide set forth in SEQ IDNO:2, respectively).

ii. Bacterial Hyaluronidases

Bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1) degrade hyaluronanand, to various extents, chondroitin sulfates and dermatan sulfates.Hyaluronan lyases isolated from bacteria differ from hyaluronidases(from other sources, e.g., hyaluronoglucosaminidases, EC 3.2.1.35) bytheir mode of action. They are endo-β-N-acetylhexosaminidases thatcatalyze an elimination reaction, rather than hydrolysis, of theβ1→4-glycosidic linkage between N-acetyl-beta-D-glucosamine andD-glucuronic acid residues in hyaluronan, yielding3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine tetra- andhexasaccharides, and disaccharide end products. The reaction results inthe formation of oligosaccharides with unsaturated hexuronic acidresidues at their nonreducing ends.

Exemplary hyaluronidases from bacteria for use in the compositions,combinations and methods provided include, but are not limited to,hyaluronan degrading enzymes in microorganisms, including strains ofArthrobacter, Bdellovibrio, Clostridium, Micrococcus, Streptococcus,Peptococcus, Propionibacterium, Bacteroides, and Streptomyces.Particular examples of such strains and enzymes include, but are notlimited to Arthrobacter sp. (strain FB24) (SEQ ID NO:67), Bdellovibriobacteriovorus (SEQ ID NO:68), Propionibacterium acnes (SEQ ID NO:69),Streptococcus agalactiae ((SEQ ID NO:70); 18RS21 (SEQ ID NO:71);serotype Ia (SEQ ID NO:72); serotype III (SEQ ID NO:73), Staphylococcusaureus (strain COL (SEQ ID NO:74); strain MRSA252 (SEQ ID NOS:75 and76); strain MSSA476 (SEQ ID NO:77); strain NCTC 8325 (SEQ ID NO:78);strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300 (SEQ IDNO:81), Streptococcus pneumoniae ((SEQ ID NO:82); strain ATCC BAA-255/R6(SEQ ID NO:83); serotype 2, strain D39/NCTC 7466 (SEQ ID NO:84),Streptococcus pyogenes (serotype (SEQ ID NO:85); serotype M2, strainMGAS10270 (SEQ ID NO:86); serotype M4, strain MGAS10750 (SEQ ID NO:87);serotype M6 (SEQ ID NO:88); serotype M12, strain MGAS2096 (SEQ ID NOS:89and 90); serotype M12, strain MGAS9429 (SEQ ID NO:91); serotype M28 (SEQID NO:92); Streptococcus suis (SEQ ID NOS:93-95); Vibrio fischeri(strain ATCC 700601/ES114 (SEQ ID NO:96)), and the Streptomyceshyaluronolyticus hyaluronidase enzyme, which is specific for hyaluronicacid and does not cleave chondroitin or chondroitin sulfate (Ohya, T.and Kaneko, Y. (1970) Biochim. Biophys. Acta 198:607).

iii. Hyaluronidases from Leeches, Other Parasites and Crustaceans

Hyaluronidases from leeches, other parasites, and crustaceans (EC3.2.1.36) are endo-β-glucuronidases that generate tetra- andhexasaccharide end-products. These enzymes catalyze hydrolysis of1→3-linkages between β-D-glucuronate and N-acetyl-D-glucosamine residuesin hyaluronate. Exemplary hyaluronidases from leeches include, but arenot limited to, hyaluronidase from Hirudinidae (e.g., Hirudomedicinalis), Erpobdellidae (e.g., Nephelopsis obscura and Erpobdellapunctata), Glossiphoniidae (e.g., Desserobdella picta, Helobdellastagnalis, Glossiphonia complanata, Placobdella ornata and Theromyzonsp.) and Haemopidae (Haemopis marmorata) (Hovingh et al. (1999) CompBiochem Physiol B Biochem Mol Biol. 124(3):319-26). An exemplaryhyaluronidase from bacteria that has the same mechanism of action as theleech hyaluronidase is that from the cyanobacteria, Synechococcus sp.(strain RCC307, SEQ ID NO:97).

b. Other Hyaluronan Degrading Enzymes

In addition to the hyaluronidase family, other hyaluronan degradingenzymes can be used in the compositions, combinations and methodsprovided. For example, enzymes, including particular chondroitinases andlyases, that have the ability to cleave hyaluronan can be employed.Exemplary chondroitinases that can degrade hyaluronan include, but arenot limited to, chondroitin ABC lyase (also known as chondroitinaseABC), chondroitin AC lyase (also known as chondroitin sulfate lyase orchondroitin sulfate eliminase) and chondroitin C lyase. Methods forproduction and purification of such enzymes for use in the compositions,combinations, and methods provided are known in the art (e.g., U.S. Pat.No. 6,054,569; Yamagata, et al. (1968) J. Biol. Chem. 243(7):1523-1535;Yang et al. (1985) J. Biol. Chem. 160(30):1849-1857).

Chondroitin ABC lyase contains two enzymes, chondroitin-sulfate-ABCendolyase (EC 4.2.2.20) and chondroitin-sulfate-ABC exolyase (EC4.2.2.21) (Hamai et al. (1997) J Biol Chem. 272(14):9123-30), whichdegrade a variety of glycosaminoglycans of the chondroitin-sulfate- anddermatan-sulfate type. Chondroitin sulfate, chondroitin-sulfateproteoglycan and dermatan sulfate are the preferred substrates forchondroitin-sulfate-ABC endolyase, but the enzyme also can act onhyaluronan at a lower rate. Chondroitin-sulfate-ABC endolyase degrades avariety of glycosaminoglycans of the chondroitin-sulfate- anddermatan-sulfate type, producing a mixture of Δ4-unsaturatedoligosaccharides of different sizes that are ultimately degraded toΔ4-unsaturated tetra- and disaccharides. Chondroitin-sulfate-ABCexolyase has the same substrate specificity but removes disaccharideresidues from the non-reducing ends of both polymeric chondroitinsulfates and their oligosaccharide fragments produced bychondroitin-sulfate-ABC endolyase (Hamai, A. et al. (1997) J. Biol.Chem. 272:9123-9130). Exemplary chondroitin-sulfate-ABC endolyases andchondroitin-sulfate-ABC exolyases include, but are not limited to, thosefrom Proteus vulgaris and Flavobacterium heparinum (the Proteus vulgarischondroitin-sulfate-ABC endolyase is set forth in SEQ ID NO: 98 (Sato etal. (1994) Appl. Microbiol. Biotechnol. 41(1):39-46).

Chondroitin AC lyase (EC 4.2.2.5) is active on chondroitin sulfates Aand C, chondroitin and hyaluronic acid, but is not active on dermatansulfate (chondroitin sulfate B). Exemplary chondroitinase AC enzymesfrom the bacteria include, but are not limited to, those fromFlavobacterium heparinum and Victivallis vadensis, set forth in SEQ IDNOS:99 and 100, respectively, and Arthrobacter aurescens (Tkalec et al.(2000) Applied and Environmental Microbiology 66(1):29-35; Ernst et al.(1995) Critical Reviews in Biochemistry and Molecular Biology30(5):387-444).

Chondroitinase C cleaves chondroitin sulfate C producing tetrasaccharideplus an unsaturated 6-sulfated disaccharide (delta Di-6S). It alsocleaves hyaluronic acid producing unsaturated non-sulfated disaccharide(delta Di-OS). Exemplary chondroitinase C enzymes from the bacteriainclude, but are not limited to, those from Streptococcus andFlavobacterium (Hibi et al. (1989) FEMS-Microbiol-Lett. 48(2):121-4;Michelacci et al. (1976) J. Biol. Chem. 251:1154-8; Tsuda et al. (1999)Eur. J. Biochem. 262:127-133)

c. Soluble Hyaluronan Degrading Enzymes

Provided in the compositions, combinations, uses and methods herein aresoluble hyaluronan degrading enzymes, including soluble hyaluronidases.Soluble hyaluronan degrading enzymes include any hyaluronan degradingenzymes that are secreted from cells (e.g. CHO cell) upon expression andexist in soluble form. Such enzymes include, but are not limited to,soluble hyaluronidases, including non-human soluble hyaluronidases,including non-human animal soluble hyaluronidases, bacterial solublehyaluronidases and human hyaluronidases, Hyal1, bovine PH20 and ovinePH20, allelic variants thereof and other variants thereof. For example,included among soluble hyaluronan degrading enzymes are any hyaluronandegrading enzymes that have been modified to be soluble. For example,hyaluronan degrading enzymes that contain a GPI anchor can be madesoluble by truncation of and removal of all or a portion of the GPIanchor. In one example, the human hyaluronidase PH20, which is normallymembrane anchored via a GPI anchor, can be made soluble by truncation ofand removal of all or a portion of the GPI anchor at the C-terminus.

Soluble hyaluronan degrading enzymes also include neutral active andacid active hyaluronidases. Depending on factors, such as, but notlimited to, the desired level of activity of the enzyme followingadministration and/or site of administration, neutral active and acidactive hyaluronidases can be selected. In a particular example, thehyaluronan degrading enzyme for use in the compositions, combinationsand methods herein is a soluble neutral active hyaluronidase.

Exemplary of a soluble hyaluronidase is PH20 from any species, such asany set forth in any of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63-65 and101-102, or truncated forms thereof lacking all or a portion of theC-terminal GPI anchor, so long as the hyaluronidase is soluble (secretedupon expression) and retains hyaluronidase activity. Also included amongsoluble hyaluronidases are allelic variants or other variants of any ofSEQ ID NOS:1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, or truncatedforms thereof. Allelic variants and other variants are known to one ofskill in the art, and include polypeptides having 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toany of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, ortruncated forms thereof. Amino acid variants include conservative andnon-conservative mutations. It is understood that residues that areimportant or otherwise required for the activity of a hyaluronidase,such as any described above or known to skill in the art, are generallyinvariant and cannot be changed. These include, for example, active siteresidues. Thus, for example, amino acid residues 111, 113 and 176(corresponding to residues in the mature PH20 polypeptide set forth inSEQ ID NO:2) of a human PH20 polypeptide, or soluble form thereof, aregenerally invariant and are not altered. Other residues that conferglycosylation and formation of disulfide bonds required for properfolding also can be invariant.

In some instances, the soluble hyaluronan degrading enzyme is normallyGPI-anchored (such as, for example, human PH20) and is rendered solubleby truncation at the C-terminus. Such truncation can remove all of theGPI anchor attachment signal sequence, or can remove only some of theGPI anchor attachment signal sequence. The resulting polypeptide,however, is soluble. In instances where the soluble hyaluronan degradingenzyme retains a portion of the GPI anchor attachment signal sequence,1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI-anchorattachment signal sequence can be retained, provided the polypeptide issoluble. Polypeptides containing one or more amino acids of the GPIanchor are termed extended soluble hyaluronan degrading enzymes. One ofskill in the art can determine whether a polypeptide is GPI-anchoredusing methods well known in the art. Such methods include, but are notlimited to, using known algorithms to predict the presence and locationof the GPI-anchor attachment signal sequence and co-site, and performingsolubility analyses before and after digestion withphosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

Extended soluble hyaluronan degrading enzymes can be produced by makingC-terminal truncations to any naturally GPI-anchored hyaluronandegrading enzyme such that the resulting polypeptide is soluble andcontains one or more amino acid residues from the GPI-anchor attachmentsignal sequence (see, e.g., U.S. Published Pat. Appl. No.US20100143457). Exemplary extended soluble hyaluronan degrading enzymesthat are C-terminally truncated but retain a portion of the GPI anchorattachment signal sequence include, but are not limited to, extendedsoluble PH20 (esPH20) polypeptides of primate origin, such as, forexample, human and chimpanzee esPH20 polypeptides. For example, theesPH20 polypeptides can be made by C-terminal truncation of any of themature or precursor polypeptides set forth in SEQ ID NOS:1, 2 or 101, orallelic or other variation thereof, including active fragment thereof,wherein the resulting polypeptide is soluble and retains one or moreamino acid residues from the GPI-anchor attachment signal sequence.Allelic variants and other variants are known to one of skill in theart, and include polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95% or more sequence identity to any of SEQ ID NOS: 1 or 2. TheesPH20 polypeptides provided herein can be C-terminally truncated by 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to the wild typepolypeptide, such as a polypeptide with a sequence set forth in SEQ IDNOS: 1, 2 or 101, provided the resulting esPH20 polypeptide is solubleand retains 1 or more amino acid residues from the GPI-anchor attachmentsignal sequence.

Typically, for use in the compositions, combinations and methods herein,a soluble human hylauronan degrading enzyme, such as a soluble humanPH20, is used. Although hylauronan degrading enzymes, such as PH20, fromother animals can be utilized, such preparations are potentiallyimmunogenic, since they are animal proteins. For example, a significantproportion of patients demonstrate prior sensitization secondary toingested foods, and since these are animal proteins, all patients have arisk of subsequent sensitization. Thus, non-human preparations may notbe suitable for chronic use. If non-human preparations are desired, itis contemplated herein that such polypeptides can be prepared to havereduced immunogenicity. Such modifications are within the level of oneof skill in the art and can include, for example, removal and/orreplacement of one or more antigenic epitopes on the molecule.

Hyaluronan degrading enzymes, including hyaluronidases (e.g., PH20),used in the methods herein can be recombinantly produced or can bepurified or partially-purified from natural sources, such as, forexample, from testes extracts. Methods for production of recombinantproteins, including recombinant hyaluronan degrading enzymes, areprovided elsewhere herein and are well known in the art.

i. Soluble Human PH20

Exemplary of a soluble hyaluronidase is soluble human PH20, Solubleforms of recombinant human PH20 have been produced and can be used inthe compositions, combinations and methods described herein. Theproduction of such soluble forms of PH20 is described in U.S. PublishedPatent Application Nos. US20040268425; US20050260186, US20060104968,US20100143457 and International PCT application No. WO2009111066. Forexample, soluble PH20 polypeptides, include C-terminally truncatedvariant polypeptides that include a sequence of amino acids in SEQ IDNO:1, or have at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98% sequenceidentity to a sequence of amino acids included in SEQ ID NO:1, retainhyaluronidase activity and are soluble. Included among thesepolypeptides are soluble PH20 polypeptides that completely lack all or aportion of the GPI-anchor attachment signal sequence.

Also included are extended soluble PH20 (esPH20) polypeptides thatcontain at least one amino acid of the GPI anchor. Thus, instead ofhaving a GPI-anchor covalently attached to the C-terminus of the proteinin the ER and being anchored to the extracellular leaflet of the plasmamembrane, these polypeptides are secreted and are soluble. C-terminallytruncated PH20 polypeptides can be C-terminally truncated by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60 or more amino acids compared to the full length wildtype polypeptide, such as a full length wild type polypeptide with asequence set forth in SEQ ID NOS:1 or 2, or allelic or species variantsor other variants thereof.

For example, soluble forms include, but are not limited to, C-terminaltruncated polypeptides of human PH20 set forth in SEQ ID NO:1 having aC-terminal amino acid residue 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482 and 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of thesequence of amino acids set forth in SEQ ID NO:1, or polypeptides thatexhibit at least 85% identity thereto. Soluble forms of human PH20generally include those that contain amino acids 36-464 set forth in SEQID NO:1. For example, when expressed in mammalian cells, the 35 aminoacid N-terminal signal sequence is cleaved during processing, and themature form of the protein is secreted. Thus, the mature solublepolypeptides contain amino acids 36 to 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO:1.Table 3 provides non-limiting examples of exemplary C-terminallytruncated PH20 polypeptides, including C-terminally truncated solublePH20 polypeptides. In Table 3 below, the length (in amino acids) of theprecursor and mature polypeptides, and the sequence identifier (SEQ IDNO) in which exemplary amino acid sequences of the precursor and maturepolypeptides of the C-terminally truncated PH20 proteins are set forth,are provided. The wild-type PH20 polypeptide also is included in Table 3for comparison. In particular, exemplary of soluble hyaluronidases aresoluble human PH20 polypeptides that are 442, 443, 444, 445, 446 or 447amino acids in length, such as set forth in any of SEQ ID NOS: 4-9, orallelic or species variants or other variants thereof.

TABLE 3 Exemplary C-terminally truncated PH20 polypeptides PrecursorMature Poly- (amino Precursor (amino Mature peptide acids) SEQ ID NOacids) SEQ ID NO wildtype 509 1 474 2 SPAM1-SILF 500 139 465 183SPAM-VSIL 499 106 464 150 SPAM1-IVSI 498 140 463 184 SPAM1-FIVS 497 107462 151 SPAM1-MFIV 496 141 461 185 SPAM1-TMFI 495 108 460 152 SPAM1-ATMF494 142 459 186 SPAM1-SATM 493 109 458 153 SPAM1-LSAT 492 143 457 187SPAM1-TLSA 491 110 456 154 SPAM1-PSTL 489 111 454 155 SPAM1-SPST 488 144453 188 SPAM1-STLS 490 112 455 156 SPAM1-ASPS 487 113 452 157 SPAM1-NASP486 145 451 189 SPAM1-YNAS 485 114 450 158 SPAM1-FYNA 484 115 449 159SPAM1-IFYN 483 46 448 48 SPAM1-QIFY 482 3 447 4 SPAM1-PQIF 481 45 446 5SPAM1-EPQI 480 44 445 6 SPAM1-EEPQ 479 43 444 7 SPAM1-TEEP 478 42 443 8SPAM1-ETEE 477 41 442 9 SPAM1-METE 476 116 441 160 SPAM1-PMET 475 117440 161 SPAM1-PPME 474 118 439 162 SPAM1-KPPM 473 119 438 163 SPAM1-LKPP472 120 437 164 SPAM1-FLKP 471 121 436 165 SPAM1-AFLK 470 122 435 166SPAM1-DAFL 469 123 434 167 SPAM1-IDAF 468 124 433 168 SPAM1-CIDA 467 40432 47 SPAM1-VCID 466 125 431 169 SPAM1-GVCI 465 126 430 170

Generally soluble forms of PH20 are produced using protein expressionsystems that facilitate correct N-glycosylation to ensure thepolypeptide retains activity, since glycosylation is important for thecatalytic activity and stability of hyaluronidases. Such cells include,for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

ii. rHuPH20

Recombinant soluble forms of human PH20 have been generated and can beused in the compositions, combinations and methods provided herein. Thegeneration of such soluble forms of recombinant human PH20 aredescribed, for example, in U.S. Published Patent Application Nos.US20040268425; US 20050260186; US20060104968; US20100143457; andInternational PCT Appl. No. WO2009111066. Exemplary of such polypeptidesare those generated by expression of a nucleic acid molecule encodingamino acids 1-482 (set forth in SEQ ID NO:3). Such an exemplary nucleicacid molecule is set forth in SEQ ID NO:49. Post translationalprocessing removes the 35 amino acid signal sequence, leaving a 447amino acid soluble recombinant human PH20 (SEQ ID NO:4). As produced inthe culture medium there is heterogeneity at the C-terminus such thatthe product, designated rHuPH20, includes a mixture of species that caninclude any one or more of SEQ ID NOS. 4-9 in various abundance.Typically, rHuPH20 is produced in cells that facilitate correctN-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHOcells).

d. Glycosylation of Hyaluronan Degrading Enzymes

Glycosylation, including N- and O-linked glycosylation, of somehyaluronan degrading enzymes, including hyaluronidases, can be importantfor their catalytic activity and stability. While altering the type ofglycan modifying a glycoprotein can have dramatic affects on a protein'santigenicity, structural folding, solubility, and stability, mostenzymes are not thought to require glycosylation for optimal enzymeactivity. For some hyaluronidases, removal of N-linked glycosylation canresult in near complete inactivation of the hyaluronidase activity.Thus, for such hyaluronidases, the presence of N-linked glycans iscritical for generating an active enzyme.

N-linked oligosaccharides fall into several major types (oligomannose,complex, hybrid, sulfated), all of which have (Man)3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residuesthat fall within -Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro).Glycosylation at an -Asn-Xaa-Cys-site has been reported for coagulationprotein C. In some instances, a hyaluronan degrading enzyme, such as ahyaluronidase, can contain both N-glycosidic and O-glycosidic linkages.For example, PH20 has O-linked oligosaccharides as well as N-linkedoligosaccharides. There are seven potential N-linked glycosylation sitesat N82, N166, N235, N254, N368, N393, N490 of human PH20 exemplified inSEQ ID NO: 1. Amino acid residues N82, N166 and N254 are occupied bycomplex type glycans whereas amino acid residues N368 and N393 areoccupied by high mannose type glycans. Amino acid residue N235 isoccupied by approximately 80% high mannose type glycans and 20% complextype glycans. As noted above, N-linked glycosylation at N490 is notrequired for hyaluronidase activity.

In some examples, the hyaluronan degrading enzymes for use in thecompositions, combinations and/or methods provided are glycosylated atone or all of the glycosylation sites. For example, for human PH20, or asoluble form thereof, 2, 3, 4, 5, or 6 of the N-glycosylation sitescorresponding to amino acids N82, N166, N235, N254, N368, and N393 ofSEQ ID NO: 1 are glycosylated. In some examples the hyaluronan degradingenzymes are glycosylated at one or more native glycosylation sites. Inother examples, the hyaluronan degrading enzymes are modified at one ormore non-native glycosylation sites to confer glycosylation of thepolypeptide at one or more additional site. In such examples, attachmentof additional sugar moieties can enhance the pharmacokinetic propertiesof the molecule, such as improved half-life and/or improved activity.

In other examples, the hyaluronan degrading enzymes for use in thecompositions, combinations and/or methods provided herein are partiallydeglycosylated (or N-partially glycosylated polypeptides). For example,partially deglycosylated soluble PH20 polypeptides that retain all or aportion of the hyaluronidase activity of a fully glycosylatedhyaluronidase can be used in the compositions, combinations and/ormethods provided herein. Exemplary partially deglycosylatedhyalurodinases include soluble forms of a partially deglycosylated PH20polypeptides from any species, such as any set forth in any of SEQ IDNOS: 1, 2, 11, 25, 27, 29-32, 63, 65, and 101-102, or allelic variants,truncated variants, or other variants thereof. Such variants are knownto one of skill in the art, and include polypeptides having 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95% or more sequence identity to any ofSEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63, 65, and 101-102, or truncatedforms thereof. The partially deglycosylated hyaluronidases providedherein also include hybrid, fusion and chimeric partially deglycosylatedhyaluronidases, and partially deglycosylated hyaluronidase conjugates.

Glycosidases, or glycoside hydrolases, are enzymes that catalyze thehydrolysis of the glycosidic linkage to generate two smaller sugars. Themajor types of N-glycans in vertebrates include high mannose glycans,hybrid glycans and complex glycans. There are several glycosidases thatresult in only partial protein deglycosylation, including: EndoF1, whichcleaves high mannose and hybrid type glycans; EndoF2, which cleavesbiantennary complex type glycans; EndoF3, which cleaves biantennary andmore branched complex glycans; and EndoH, which cleaves high mannose andhybrid type glycans. Treatment of a hyaluronan degrading enzyme, such asa soluble hyaluronidase, such as a soluble PH20, with one or all ofthese glycosidases can result in only partial deglycosylation and,therefore, retention of hyaluronidase activity.

Partially deglycosylated hyaluronan degrading enzymes, such as partiallydeglycosylated soluble hyaluronidases, can be produced by digestion withone or more glycosidases, generally a glycosidase that does not removeall N-glycans but only partially deglycosylates the protein. Forexample, treatment of PH20 (e.g. a recombinant PH20 designated rHuPH20)with one or all of the above glycosidases (e.g. EndoF1, EndoF2 and/orEndoF3) results in partial deglycosylation. These partiallydeglycosylated PH20 polypeptides can exhibit hyaluronidase enzymaticactivity that is comparable to the fully glycosylated polypeptides. Incontrast, treatment of PH20 with PNGaseF, a glycosidase that cleaves allN-glycans, results in complete removal of all N-glycans and therebyrenders PH20 enzymatically inactive. Thus, although all N-linkedglycosylation sites (such as, for example, those at amino acids N82,N166, N235, N254, N368, and N393 of human PH20, exemplified in SEQ IDNO: 1) can be glycosylated, treatment with one or more glycosidases canrender the extent of glycosylation reduced compared to a hyaluronidasethat is not digested with one or more glycosidases.

The partially deglycosylated hyaluronan degrading enzymes, includingpartially deglycosylated soluble PH20 polypeptides, can have 10%, 20%,30%, 40%, 50%, 60%, 70% or 80% of the level of glycosylation of a fullyglycosylated polypeptide. In one example, 1, 2, 3, 4, 5 or 6 of theN-glycosylation sites corresponding to amino acids N82, N166, N235,N254, N368, and N393 of SEQ ID NO:1 are partially deglycosylated, suchthat they no longer contain high mannose or complex type glycans, butrather contain at least an N-acetylglucosamine moiety. In some examples,1, 2 or 3 of the N-glycosylation sites corresponding to amino acids N82,N166 and N254 of SEQ ID NO:1 are deglycosylated, that is, they do notcontain a sugar moiety. In other examples, 3, 4, 5, or 6 of theN-glycosylation sites corresponding to amino acids N82, N166, N235,N254, N368, and N393 of SEQ ID NO:1 are glycosylated. Glycosylated aminoacid residues minimally contain an N-acetylglucosamine moiety.Typically, the partially deglyclosylated hyaluronan degrading enzymes,including partially deglycosylated soluble PH20 polypeptides, exhibithyaluronidase activity that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000%or more of the hyaluronidase activity exhibited by the fullyglycosylated polypeptide.

e. Modified (Polymer-Conjugated) Hyaluronan Degrading Enzymes

In one example, the provided compositions and combinations containhyaluronan degrading enzymes, in particular soluble hyaluronidases, thathave been modified by conjugation to one or more polymeric molecule(polymer), typically to increase the half-life of the hyaluronandegrading enzyme, for example, to promote prolonged/sustained treatmenteffects in a subject.

Covalent or other stable attachment (conjugation) of polymericmolecules, such as polyethylene glycol (PEGylation moiety (PEG)), to thehyaluronan degrading enzymes, such as hyaluronidases, impart beneficialproperties to the resulting hyaluronan degrading enzyme-polymercomposition. Such properties include improved biocompatibility,extension of protein (and enzymatic activity) half-life in the blood,cells and/or in other tissues within a subject, effective shielding ofthe protein from proteases and hydrolysis, improved biodistribution,enhanced pharmacokinetics and/or pharmacodynamics, and increased watersolubility.

Exemplary polymers that can be conjugated to the hyaluronan degradingenzyme, such as the hyaluronidase, include natural and synthetichomopolymers, such as polyols (i.e. poly-OH), polyamines (i.e. poly-NH₂)and polycarboxyl acids (i.e. poly-COOH), and further heteropolymers i.e.polymers comprising one or more different coupling groups e.g. ahydroxyl group and amine groups. Examples of suitable polymericmolecules include polymeric molecules selected from among polyalkyleneoxides (PAO), such as polyalkylene glycols (PAG), including polyethyleneglycols (PEG), methoxypolyethylene glycols (mPEG) and polypropyleneglycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole(CDI-PEG) branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA),polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, dextrans including carboxymethyl-dextrans, heparin,homologous albumin, celluloses, including methylcellulose,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulosecarboxyethylcellulose and hydroxypropylcellulose, hydrolysates ofchitosan, starches such as hydroxyethyl-starches andhydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guargum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and bio-polymers.

Typically, the polymers are polyalkylene oxides (PAO), such aspolyethylene oxides, such as PEG, typically mPEG, which, in comparisonto polysaccharides such as dextran and pullulan, have few reactivegroups capable of cross-linking. Typically, the polymers are non-toxicpolymeric molecules such as (m)polyethylene glycol (mPEG) which can becovalently conjugated to the hyaluronan degrading enzyme, such as thehyaluronidase (e.g. to attachment groups on the protein's surface) usinga relatively simple chemistry.

PEGylation of therapeutics has been reported to increase resistance toproteolysis, increase plasma half-life, and decrease antigenicity andimmunogenicity. Examples of PEGylation methodologies are known in theart (see for example, Lu and Felix, Int. J. Peptide Protein Res.,43:127-138, 1994; Lu and Felix, Peptide Res., 6.142-6, 1993; Felix etal., Int. J Peptide Res., 46:253-64, 1995; Benhar et al., J. Biol.Chem., 269: 13398-404, 1994; Brumeanu et al., J ImmunoL, 154:3088-95,1995; see also, Caliceti et al. (2003) Adv. Drug Deliv. Rev.55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt 2):3S-8S).PEGylation also can be used in the delivery of nucleic acid molecules invivo. For example, PEGylation of adenovirus can increase stability andgene transfer (see, e.g., Cheng et al. (2003) Pharm. Res.20(9):1444-51).

Suitable polymeric molecules for attachment to the hyaluronan degradingenzymes, including hyaluronidases, include, but are not limited to,polyethylene glycol (PEG) and PEG derivatives such asmethoxy-polyethylene glycols (mPEG), PEG-glycidyl ethers (Epox-PEG),PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, and polyethyleneoxide (PEO) (see e.g. Roberts et al., Advanced Drug Delivery Review(2002) 54: 459-476; Harris and Zalipsky, S (eds.) “Poly(ethyleneglycol), Chemistry and Biological Applications” ACS Symposium Series680, 1997; Mehvar et al., J. Pharm. Pharmaceut. Sci., 3(1):125-136,2000; Harris, (2003) Nature Reviews Drug Discovery 2:214-221; andTsubery, (2004) J Biol. Chem 279(37):38118-24). The polymeric moleculecan be of a molecular weight typically ranging from about 3 kDa to about60 kDa. In some embodiments the polymeric molecule that is conjugated toa protein, such as rHuPH20, has a molecular weight of 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60 or more than 60 kDa.

PEGylated Soluble Hyaluronan Degrading Enzymes

The hyaluronan degrading enzyme used in the methods, compositions andcombinations herein can be a PEGylated hyaluronan degrading enzyme, suchas a PEGylated soluble hyaluronan degrading enzyme. In one example, itis a PEGylated soluble hyaluronidase, e.g. PEGylated rHuPH20. Variousmethods of modifying polypeptides by covalently attaching (conjugating)a PEG or PEG derivative (i.e. “PEGylation”) are known in the art (seee.g., U.S. 2006/0104968; U.S. Pat. No. 5,672,662; U.S. Pat. No.6,737,505; and U.S. 2004/0235734). Techniques for PEGylation include,but are not limited to, specialized linkers and coupling chemistries(see e.g., Roberts, Adv. Drug Deliv. Rev. 54:459-476, 2002), attachmentof multiple PEG moieties to a single conjugation site (such as via useof branched PEGs; see e.g., Guiotto et al., Bioorg. Med. Chem. Lett.12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation (seee.g., Chapman et al., Nature Biotech. 17:780-783, 1999), andsite-directed enzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv.Rev., 54:487-504, 2002). Methods and techniques described in the art canproduce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10PEG or PEG derivatives attached to a single protein molecule (see e.g.,U.S. 2006/0104968).

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG₂-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutryaldehyde, branched mPEG₂ butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardiniet al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. BioactiveCompatible Polymers 12:197-207, 1997; U.S. Pat. No. 5,672,662; U.S. Pat.No. 5,932,462; U.S. Pat. No. 6,495,659; U.S. Pat. No. 6,737,505; U.S.Pat. No. 4,002,531; U.S. Pat. No. 4,179,337; U.S. Pat. No. 5,122,614;U.S. 5,324, 844; U.S. Pat. No. 5,446,090; U.S. Pat. No. 5,612,460; U.S.Pat. No. 5,643,575; U.S. Pat. No. 5,766,581; U.S. Pat. No. 5,795,569;U.S. Pat. No. 5,808,096; U.S. Pat. No. 5,900,461; U.S. Pat. No.5,919,455; U.S. Pat. No. 5,985,263; U.S. Pat. No. 5,990,237; U.S. Pat.No. 6,113,906; U.S. Pat. No. 6,214,966; U.S. Pat. No. 6,258,351; U.S.Pat. No. 6,340,742; U.S. Pat. No. 6,413,507; U.S. Pat. No. 6,420,339;U.S. Pat. No. 6,437,025; U.S. Pat. No. 6,448,369; U.S. Pat. No.6,461,802; U.S. Pat. No. 6,828,401; U.S. Pat. No. 6,858,736; U.S.2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430;U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637;US 2004/0235734; WO0500360; U.S. 2005/0114037; U.S. 2005/0171328; U.S.2005/0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO0249673; WO 9428024; and WO 0187925).

D. Methods of Producing Nucleic Acids and Encoded Polypeptides ofHyaluronan Degrading Enzymes

Polypeptides of a hyaluronan degrading enzyme, such as a solublehyaluronidase, set forth herein, can be obtained by methods well knownin the art for protein purification and recombinant protein expression.Any method known to those of skill in the art for identification ofnucleic acids that encode desired genes can be used. Any methodavailable in the art can be used to obtain a full length (i.e.,encompassing the entire coding region) cDNA or genomic DNA cloneencoding a hyaluronidase, such as from a cell or tissue source. Modifiedor variant soluble hyaluronidases, can be engineered from a wildtypepolypeptide, such as by site-directed mutagenesis.

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. A nucleic acidcontaining material can be used as a starting material from which adesired polypeptide-encoding nucleic acid molecule can be isolated. Forexample, DNA and mRNA preparations, cell extracts, tissue extracts,fluid samples (e.g. blood, serum, saliva), samples from healthy and/ordiseased subjects can be used in amplification methods. Nucleic acidlibraries also can be used as a source of starting material. Primers canbe designed to amplify a desired polypeptide. For example, primers canbe designed based on expressed sequences from which a desiredpolypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. Nucleic acidmolecules generated by amplification can be sequenced and confirmed toencode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. Additional nucleotide residuessequences such as sequences of bases specifying protein binding regionsalso can be linked to enzyme-encoding nucleic acid molecules. Suchregions include, but are not limited to, sequences of residues thatfacilitate or encode proteins that facilitate uptake of an enzyme intospecific target cells, or otherwise alter pharmacokinetics of a productof a synthetic gene. For example, enzymes can be linked to PEG moieties.

In addition, tags or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residues sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Exemplary of such sequencesinclude nucleic acid sequences encoding a His tag (e.g., 6×His, HHHHHH;SEQ ID NO:54) or Flag Tag (DYKDDDDK; SEQ ID NO:55).

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). Other expression vectors include theHZ24 expression vector exemplified herein. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini.Insertion can be effected using TOPO cloning vectors (Invitrogen,Carlsbad, Calif.). If the complementary restriction sites used tofragment the DNA are not present in the cloning vector, the ends of theDNA molecules can be enzymatically modified. Alternatively, any sitedesired can be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers can contain specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andprotein gene can be modified by homopolymeric tailing. Recombinantmolecules can be introduced into host cells via, for example,transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

1. Vectors and Cells

For recombinant expression of one or more of the desired proteins, suchas any hyaluronan degrading enzyme polypeptide described herein, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the protein can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein coding sequence.The necessary transcriptional and translational signals also can besupplied by the native promoter for enzyme genes, and/or their flankingregions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, Archea, plant cells, insect cells and animalcells. The cells are used to produce a protein thereof by growing theabove-described cells under conditions whereby the encoded protein isexpressed by the cell, and recovering the expressed protein. Forpurposes herein, for example, the enzyme can be secreted into themedium.

Provided are vectors that contain a sequence of nucleotides that encodesthe hyaluronan degrading enzyme polypeptide, in some examples a solublehyaluronidase polypeptide, coupled to the native or heterologous signalsequence, as well as multiple copies thereof. The vectors can beselected for expression of the enzyme protein in the cell or such thatthe enzyme protein is expressed as a secreted protein.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”:in Scientific American 242:79-94 (1980); plant expression vectorscontaining the nopaline synthetase promoter (Herrara-Estrella et al.,Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., Nucleic Acids Res. 9:2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., Genes and Devel. 1:161-171 (1987)), beta globin gene controlregion which is active in myeloid cells (Magram et al., Nature315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., Cell 48:703-712 (1987)), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani,Nature 314:283-286 (1985)), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pQEexpression vectors (available from Qiagen, Valencia, Calif.; see alsoliterature published by Qiagen describing the system). pQE vectors havea phage T5 promoter (recognized by E. coli RNA polymerase) and a doublelac operator repression module to provide tightly regulated, high-levelexpression of recombinant proteins in E. coli, a synthetic ribosomalbinding site (RBS II) for efficient translation, a 6×His tag codingsequence, t₀ and T1 transcriptional terminators, ColE1 origin ofreplication, and a beta-lactamase gene for conferring ampicillinresistance. The pQE vectors enable placement of a 6×His tag at eitherthe N- or C-terminus of the recombinant protein. Such plasmids includepQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for allthree reading frames and provide for the expression of N-terminally6×His-tagged proteins. Other exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pETexpression vectors (see, U.S. Pat. No. 4,952,496; available fromNovagen, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a-c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET19b (Novagen, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Exemplary of a vector for mammalian cell expression is the HZ24expression vector. The HZ24 expression vector was derived from the pCIvector backbone (Promega). It contains DNA encoding the Beta-lactamaseresistance gene (AmpR), an F1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), and an SV40 latepolyadenylation signal (SV40). The expression vector also has aninternal ribosome entry site (IRES) from the ECMV virus (Clontech) andthe mouse dihydrofolate reductase (DHFR) gene.

2. Expression

Hyaluronan degrading enzyme polypeptides, including solublehyaluronidase polypeptides, can be produced by any method known to thoseof skill in the art including in vivo and in vitro methods. Desiredproteins can be expressed in any organism suitable to produce therequired amounts and forms of the proteins, such as for example, neededfor administration and treatment. Expression hosts include prokaryoticand eukaryotic organisms such as E. coli, yeast, plants, insect cells,mammalian cells, including human cell lines and transgenic animals.Expression hosts can differ in their protein production levels as wellas the types of post-translational modifications that are present on theexpressed proteins. The choice of expression host can be made based onthese and other factors, such as regulatory and safety considerations,production costs and the need and methods for purification.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoter's andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Hyaluronan degrading enzyme polypeptides, such as soluble hyaluronidasepolypeptides, also can be utilized or expressed as protein fusions. Forexample, an enzyme fusion can be generated to add additionalfunctionality to an enzyme. Examples of enzyme fusion proteins include,but are not limited to, fusions of a signal sequence, a tag such as forlocalization, e.g. a his₆ tag or a myc tag, or a tag for purification,for example, a GST fusion, and a sequence for directing proteinsecretion and/or membrane association.

a. Prokaryotic Cells

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is a simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters, such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated XPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreitol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach is theexpression of proteins in the periplasmic space of bacteria whichprovides an oxidizing environment and chaperonin-like and disulfideisomerases and can lead to the production of soluble protein. Typically,a leader sequence is fused to the protein to be expressed which directsthe protein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GAL7and GAL5 and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoter. Expression vectors often include aselectable marker such as LEU2, TRP1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as Bip and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

c. Insect Cells

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides such as hyaluronidase polypeptides. Insect cellsexpress high levels of protein and are capable of most of thepost-translational modifications used by higher eukaryotes. Baculovirushave a restrictive host range which improves the safety and reducesregulatory concerns of eukaryotic expression. Typical expression vectorsuse a promoter for high level expression such as the polyhedrin promoterof baculovirus. Commonly used baculovirus systems include thebaculoviruses such as Autographa californica nuclear polyhedrosis virus(AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and aninsect cell line such as Sf9 derived from Spodoptera frugiperda,Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-levelexpression, the nucleotide sequence of the molecule to be expressed isfused immediately downstream of the polyhedrin initiation codon of thevirus. Mammalian secretion signals are accurately processed in insectcells and can be used to secrete the expressed protein into the culturemedium. In addition, the cell lines Pseudaletia unipuncta (A7S) andDanaus plexippus (DpN1) produce proteins with glycosylation patternssimilar to mammalian cell systems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schneider 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Cells

Mammalian expression systems can be used to express proteins includinghyaluronan degrading enzyme polypeptides, such as soluble hyaluronidasepolypeptides. Expression constructs can be transferred to mammaliancells by viral infection such as adenovirus or by direct DNA transfersuch as liposomes, calcium phosphate, DEAE-dextran and by physical meanssuch as electroporation and microinjection. Expression vectors formammalian cells typically include an mRNA cap site, a TATA box, atranslational initiation sequence (Kozak consensus sequence) andpolyadenylation elements. IRES elements also can be added to permitbicistronic expression with another gene, such as a selectable marker.Such vectors often include transcriptional promoter-enhancers forhigh-level expression, for example the SV40 promoter-enhancer, the humancytomegalovirus (CMV) promoter and the long terminal repeat of Roussarcoma virus (RSV). These promoter-enhancers are active in many celltypes. Tissue and cell-type promoters and enhancer regions also can beused for expression. Exemplary promoter/enhancer regions include, butare not limited to, those from genes such as elastase I, insulin,immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein,alpha 1 antitrypsin, beta globin, myelin basic protein, myosin lightchain 2, and gonadotropic releasing hormone gene control. Selectablemarkers can be used to select for and maintain cells with the expressionconstruct. Examples of selectable marker genes include, but are notlimited to, hygromycin B phosphotransferase, adenosine deaminase,xanthine-guanine phosphoribosyl transferase, aminoglycosidephosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase.For example, expression can be performed in the presence of methotrexateto select for only those cells expressing the DHFR gene. Fusion withcell surface signaling molecules such as TCR-ζ and Fc_(ε)RI-γ can directexpression of the proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NSO(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media which facilitates purification of secretedproteins from the cell culture media. Examples include CHO-S cells(Invitrogen, Carlsbad, Calif., cat #11619-012) and the serum free EBNA-1cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-342). Celllines also are available that are adapted to grow in special mediaoptimized for maximal expression. For example, DG44 CHO cells areadapted to grow in suspension culture in a chemically defined, animalproduct-free medium.

e. Plants

Transgenic plant cells and plants can be used to express proteins suchas any described herein. Expression constructs are typically transferredto plants using direct DNA transfer such as microprojectile bombardmentand PEG-mediated transfer into protoplasts, and withagrobacterium-mediated transformation. Expression vectors can includepromoter and enhancer sequences, transcriptional termination elementsand translational control elements. Expression vectors andtransformation techniques are usually divided between dicot hosts, suchas Arabidopsis and tobacco, and monocot hosts, such as corn and rice.Examples of plant promoters used for expression include the cauliflowermosaic virus promoter, the nopaline synthetase promoter, the ribosebisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.Selectable markers such as hygromycin, phosphomannose isomerase andneomycin phosphotransferase are often used to facilitate selection andmaintenance of transformed cells. Transformed plant cells can bemaintained in culture as cells, aggregates (callus tissue) orregenerated into whole plants. Transgenic plant cells also can includealgae engineered to produce hyaluronidase polypeptides. Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice of protein produced in these hosts.

3. Purification Techniques

Method for purification of polypeptides, including hyaluronan degradingenzyme polypeptides (e.g. soluble hyaluronidase polypeptides) or otherproteins, from host cells will depend on the chosen host cells andexpression systems. For secreted molecules, proteins are generallypurified from the culture media after removing the cells. Forintracellular expression, cells can be lysed and the proteins purifiedfrom the extract. When transgenic organisms such as transgenic plantsand animals are used for expression, tissues or organs can be used asstarting material to make a lysed cell extract. Additionally, transgenicanimal production can include the production of polypeptides in milk oreggs, which can be collected, and if necessary, the proteins can beextracted and further purified using standard methods in the art.

Proteins, such as soluble hyaluronidase polypeptides, can be purifiedusing standard protein purification techniques known in the artincluding but not limited to, SDS-PAGE, size fraction and size exclusionchromatography, ammonium sulfate precipitation and ionic exchangechromatography, such as anion exchange chromatography. Affinitypurification techniques also can be utilized to improve the efficiencyand purity of the preparations. For example, antibodies, receptors andother molecules that bind hyaluronidase enzymes can be used in affinitypurification. Expression constructs also can be engineered to add anaffinity tag to a protein such as a myc epitope, GST fusion or His₆ andaffinity purified with myc antibody, glutathione resin and Ni-resin,respectively. Purity can be assessed by any method known in the artincluding gel electrophoresis and staining and spectrophotometrictechniques. Purified rHuPH20 compositions, as provided herein, typicallyhave a specific activity of at least 70,000 to 100,000 Units/mg, forexample, about 120,000 Units/mg. The specific activity can vary uponmodification, such as with a polymer.

4. PEGylation of Hyaluronan Degrading Enzyme Polypeptides

Polyethylene glycol (PEG) has been widely used in biomaterials,biotechnology and medicine primarily because PEG is a biocompatible,nontoxic, water-soluble polymer that is typically nonimmunogenic (Zhaoand Harris, ACS Symposium Series 680: 458-72, 1997). In the area of drugdelivery, PEG derivatives have been widely used in covalent attachment(i.e., “PEGylation”) to proteins to reduce immunogenicity, proteolysisand kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del.Rev. 16:157-82, 1995). Similarly, PEG has been attached to low molecularweight, relatively hydrophobic drugs to enhance solubility, reducetoxicity and alter biodistribution. Typically, PEGylated drugs areinjected as solutions.

A closely related application is synthesis of crosslinked degradable PEGnetworks or formulations for use in drug delivery since much of the samechemistry used in design of degradable, soluble drug carriers can alsobe used in design of degradable gels (Sawhney et al., Macromolecules 26:581-87, 1993). It also is known that intermacromolecular complexes canbe formed by mixing solutions of two complementary polymers. Suchcomplexes are generally stabilized by electrostatic interactions(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) betweenthe polymers involved, and/or by hydrophobic interactions between thepolymers in an aqueous surrounding (Krupers et al., Eur. Polym J.32:785-790, 1996). For example, mixing solutions of polyacrylic acid(PAAc) and polyethylene oxide (PEO) under the proper conditions resultsin the formation of complexes based mostly on hydrogen bonding.Dissociation of these complexes at physiologic conditions has been usedfor delivery of free drugs (i.e., non-PEGylated). In addition, complexesof complementary polymers have been formed from both homopolymers andcopolymers.

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG₂-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutryaldehyde, branched mPEG₂ butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardiniet al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. BioactiveCompatible Polymers 12:197-207, 1997; U.S. Pat. No. 5,672,662; U.S. Pat.No. 5,932,462; U.S. Pat. No. 6,495,659; U.S. Pat. No. 6,737,505; U.S.Pat. No. 4,002,531; U.S. Pat. No. 4,179,337; U.S. Pat. No. 5,122,614;U.S. 5,324, 844; U.S. Pat. No. 5,446,090; U.S. Pat. No. 5,612,460; U.S.Pat. No. 5,643,575; U.S. Pat. No. 5,766,581; U.S. Pat. No. 5,795,569;U.S. Pat. No. 5,808,096; U.S. Pat. No. 5,900,461; U.S. Pat. No.5,919,455; U.S. Pat. No. 5,985,263; U.S. Pat. No. 5,990,237; U.S. Pat.No. 6,113,906; U.S. Pat. No. 6,214,966; U.S. Pat. No. 6,258,351; U.S.Pat. No. 6,340,742; U.S. Pat. No. 6,413,507; U.S. Pat. No. 6,420,339;U.S. Pat. No. 6,437,025; U.S. Pat. No. 6,448,369; U.S. Pat. No.6,461,802; U.S. Pat. No. 6,828,401; U.S. Pat. No. 6,858,736; U.S.2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430;U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637;US 2004/0235734; WO0500360; U.S. 2005/0114037; U.S. 2005/0171328; U.S.2005/0209416; EP 01064951; EP 0822199; WO 01076640; WO 0002017; WO0249673; WO 9428024; and WO 0187925).

In one example, the polyethylene glycol has a molecular weight rangingfrom about 3 kD to about 50 kD, and typically from about 5 kD to about30 kD. Covalent attachment of the PEG to the drug (known as“PEGylation”) can be accomplished by known chemical synthesistechniques. For example, the PEGylation of protein can be accomplishedby reacting NHS-activated PEG with the protein under suitable reactionconditions.

While numerous reactions have been described for PEGylation, those thatare most generally applicable confer directionality, utilize mildreaction conditions, and do not necessitate extensive downstreamprocessing to remove toxic catalysts or bi-products. For instance,monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thusits use limits some of the heterogeneity of the resulting PEG-proteinproduct mixture. Activation of the hydroxyl group at the end of thepolymer opposite to the terminal methoxy group is generally necessary toaccomplish efficient protein PEGylation, with the aim being to make thederivatised PEG more susceptible to nucleophilic attack. The attackingnucleophile is usually the epsilon-amino group of a lysyl residue, butother amines also can react (e.g. the N-terminal alpha-amine or the ringamines of histidine) if local conditions are favorable. A more directedattachment is possible in proteins containing a single lysine orcysteine. The latter residue can be targeted by PEG-maleimide forthiol-specific modification. Alternatively, PEG hydrazide can be reactedwith a periodate oxidized hyaluronan degrading enzyme and reduced in thepresence of NaCNBH₃. More specifically, PEGylated CMP sugars can bereacted with a hyaluronan degrading enzyme in the presence ofappropriate glycosyl-transferases. One technique is the “PEGylation”technique where a number of polymeric molecules are coupled to thepolypeptide in question. When using this technique the immune system hasdifficulties in recognizing the epitopes on the polypeptide's surfaceresponsible for the formation of antibodies, thereby reducing the immuneresponse. For polypeptides introduced directly into the circulatorysystem of the human body to give a particular physiological effect (i.e.pharmaceuticals) the typical potential immune response is an IgG and/orIgM response, while polypeptides which are inhaled through therespiratory system (i.e. industrial polypeptide) potentially can causean IgE response (i.e. allergic response). One of the theories explainingthe reduced immune response is that the polymeric molecule(s) shield(s)epitope(s) on the surface of the polypeptide responsible for the immuneresponse leading to antibody formation. Another theory or at least apartial factor is that the heavier the conjugate is, the more reducedimmune response is obtained.

Typically, to make the PEGylated hyaluronan degrading enzymes providedherein, including the PEGylated hyaluronidases, PEG moieties areconjugated, via covalent attachment, to the polypeptides. Techniques forPEGylation include, but are not limited to, specialized linkers andcoupling chemistries (see e.g., Roberts, Adv. Drug Deliv. Rev.54:459-476, 2002), attachment of multiple PEG moieties to a singleconjugation site (such as via use of branched PEGs; see e.g., Guiotto etal., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specificPEGylation and/or mono-PEGylation (see e.g., Chapman et al., NatureBiotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (seee.g., Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002). Methods andtechniques described in the art can produce proteins having 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives' attached to asingle protein molecule (see e.g., U.S. 2006/0104968).

As an exemplary illustration of the PEGylation of an illustrative methodfor making PEGylated hyaluronan degrading enzymes, such as PEGylatedhyaluronidases, PEG aldehydes, succinimides and carbonates have eachbeen applied to conjugate PEG moieties, typically succinimidyl PEGs, torHuPH20. For example, rHuPH20 has been conjugated with exemplarysuccinimidyl monoPEG (mPEG) reagents including mPEG-SuccinimidylPropionates (mPEG-SPA), mPEG-Succinimidyl Butanoates (mPEG-SBA), and(for attaching “branched” PEGs) mPEG2-N-Hydroxylsuccinimide. ThesePEGylated succinimidyl esters contain different length carbon backbonesbetween the PEG group and the activated cross-linker, and either asingle or branched PEG group. These differences can be used, forexample, to provide for different reaction kinetics and to potentiallyrestrict sites available for PEG attachment to rHuPH20 during theconjugation process.

Succinimidyl PEGs (as above) comprising either linear or branched PEGscan be conjugated to rHuPH20. PEGs can used to generate rHuPH20sreproducibly containing molecules having, on the average, between aboutthree to six or three to six PEG molecules per hyaluronidase. SuchPEGylated rHuPH20 compositions can be readily purified to yieldcompositions having specific activities of approximately 25,000 or30,000 Unit/mg protein hyaluronidase activity, and being substantiallyfree of non-PEGylated rHuPH20 (less than 5% non-PEGylated).

Using various PEG reagents, exemplary versions of hyaluronan degradingenzymes, in particular soluble human recombinant hyaluronidases (e.g.rHuPH20), can be prepared, for example, using mPEG-SBA (30 kD), mPEG-SMB(30 kD), and branched versions based on mPEG2-NHS (40 kD) and mPEG2-NHS(60 kD). PEGylated versions of rHuPH20 have been generated using NHSchemistries, as well as carbonates, and aldehydes, using each of thefollowing reagents: mPEG2-NHS-40K branched, mPEG-NHS-10K branched,mPEG-NHS-20K branched, mPEG2-NHS-60K branched; mPEG-SBA-5K,mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB-20K, mPEG-SMB-30K;mPEG-butyrldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin.PEGylated hyaluronidases have also been prepared using PEG reagentsavailable from Dowpharma, a division of Dow Chemical Corporation;including hyaluronidases PEGylated with Dowpharma'sp-nitrophenyl-carbonate PEG (30 kDa) and with propionaldehyde PEG (30kDa).

In one example, the PEGylation includes conjugation of mPEG-SBA, forexample, mPEG-SBA-30K (having a molecular weight of about 30 kDa) oranother succinimidyl esters of PEG butanoic acid derivative, to asoluble hyaluronidase. Succinimidyl esters of PEG butanoic acidderivatives, such as mPEG-SBA-30K readily couple to amino groups ofproteins. For example, covalent conjugation of m-PEG-SBA-30K and rHuPH20(which is approximately 60 KDa in size) provides stable amide bondsbetween rHuPH20 and mPEG, as shown in Scheme 1, below.

Typically, the mPEG-SBA-30K or other PEG is added to the hyaluronandegrading enzyme, in some instances a hyaluronidase, at aPEG:polypeptide molar ratio of 10:1 in a suitable buffer, e.g. 130 mMNaCl/10 mM HEPES at pH 6.8 or 70 mM phosphate buffer, pH 7, followed bysterilization, e.g. sterile filtration, and continued conjugation, forexample, with stirring, overnight at 4° C. in a cold room. In oneexample, the conjugated PEG-hyaluronan degrading enzyme is concentratedand buffer-exchanged.

Other methods of coupling succinimidyl esters of PEG butanoic acidderivatives, such as mPEG-SBA-30K are known in the art (see e.g., U.S.Pat. No. 5,672,662; U.S. Pat. No. 6,737,505; and U.S. 2004/0235734). Forexample, a polypeptide, such as a hyaluronan degrading enzyme (e.g. ahyaluronidase), can be coupled to an NHS activated PEG derivative byreaction in a borate buffer (0.1 M, pH 8.0) for one hour at 4° C. Theresulting PEGylated protein can be purified by ultrafiltration.Alternatively, PEGylation of a bovine alkaline phosphatase can beaccomplished by mixing the phosphatase with mPEG-SBA in a buffercontaining 0.2 M sodium phosphate and 0.5 M NaCl (pH 7.5) at 4° C. for30 minutes. Unreacted PEG can be removed by ultrafiltration. Anothermethod reacts polypeptide with mPEG-SBA in deionized water to whichtriethylamine is added to raise the pH to 7.2-9. The resulting mixtureis stirred at room temperature for several hours to complete thePEGylation.

Methods for PEGylation of hyaluronan degrading polypeptides, including,for example, animal-derived hyaluronidases and bacterial hyaluronandegrading enzymes, are known to one of skill in the art. See, forexample, European Patent No. EP 0400472, which describes the PEGylationof bovine testes hyaluorindase and chondroitin ABC lyase. Also, U.S.Publication No. 2006014968 describes PEGylation of a human hyaluronidasederived from human PH20. For example, the PEGylated hyaluronan-degradingenzyme generally contains at least 3 PEG moieties per molecule. Forexample, the hyaluronan-degrading enzyme can have a PEG to protein molarratio between 5:1 and 9:1, for example, 7:1.

E. Corticosteroids

Corticosteroids are a class of steroid hormones that are produced in theadrenal cortex. Corticosteroids are involved in a wide range ofphysiologic systems such as stress response, immune response andregulation of inflammation, carbohydrate metabolism, protein catabolism,blood electrolyte levels, and behavior. These include glucocorticoids,which are anti-inflammatory agents with a large number of otherfunctions and mineralocorticoids, which control salt and water balanceprimarily through action on the kidneys.

Glucocorticoids are a class of steroid hormones, e.g., corticosteroids,that bind to the glucocorticoid receptor. Glucocorticoids cause theireffects by binding to the glucocorticoid receptor. The activatedglucocorticoid complex in turn up-regulates the expression ofanti-inflammatory proteins in the nucleus and represses the expressionof pro-inflammatory proteins in the cytosol by preventing thetranslocation of other transcription factors from the cytosol into thenucleus.

Generally, any corticosteroid, e.g., glucocorticoid, can be used in themethods or combinations provided herein. The glucocorticoids includesynthetic and non-synthetic glucocorticoids. Exemplary glucocorticoidsinclude, but are not limited to: alclomethasones, algestones,beclomethasones (e.g. beclomethasone dipropionate), betamethasones (e.g.betamethasone 17-valerate, betamethasone sodium acetate, betamethasonesodium phosphate, betamethasone valerate), budesonides, clobetasols(e.g. clobetasol propionate), clobetasones, clocortolones (e.g.clocortolone pivalate), cloprednols, corticosterones, cortisones andhydrocortisones (e.g. hydrocortisone acetate), cortivazols,deflazacorts, desonides, desoximethasones, dexamethasones (e.g.dexamethasone 21-phosphate, dexamethasone acetate, dexamethasone sodiumphosphate), diflorasones (e.g. diflorasone diacetate), diflucortolones,difluprednates, enoxolones, fluazacorts, flucloronides, fludrocortisones(e.g., fludrocortisone acetate), flumethasones (e.g. flumethasonepivalate), flunisolides, fluocinolones (e.g. fluocinolone acetonide),fluocinonides, fluocortins, fluocortolones, fluorometholones (e.g.fluorometholone acetate), fluperolones (e.g., fluperolone acetate),fluprednidenes, fluprednisolones, flurandrenolides, fluticasones (e.g.fluticasone propionate), formocortals, halcinonides, halobetasols,halometasones, halopredones, hydrocortamates, hydrocortisones (e.g.hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisoneacetate, hydrocortisone buteprate, hydrocortisone butyrate,hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisoneprobutate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, hydrocortisone valerate), loteprednol etabonate,mazipredones, medrysones, meprednisones, methylprednisolones(methylprednisolone aceponate, methylprednisolone acetate,methylprednisolone hemisuccinate, methylprednisolone sodium succinate),mometasones (e.g., mometasone furoate), paramethasones (e.g.,paramethasone acetate), prednicarbates, prednisolones (e.g. prednisolone25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone21-hemisuccinate, prednisolone acetate; prednisolone farnesylate,prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide),prednisolone metasulphobenzoate, prednisolone steaglate, prednisolonetebutate, prednisolone tetrahydrophthalate), prednisones, prednivals,prednylidenes, rimexolones, tixocortols, triamcinolones (e.g.triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide, triamcinolone acetonide 21-palmitate, triamcinolonediacetate). These glucocorticoids and the salts thereof are discussed indetail, for example, in Remington's Pharmaceutical Sciences, A. Osol,ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980).

In some examples, the glucocorticoid is selected from among cortisones,dexamethasones, hydrocortisones, methylprednisolones, prednisolones andprednisones. In a particular example, the glucocorticoid isdexamethasone.

F. Use of Corticosteroids to Ameliorate the Adverse Effects of anAnti-Hyaluronan Agent

Provided herein are methods of treatment or uses with corticosteroids,particularly glucocorticoids, e.g., dexamethasone, orally orintravenously, to eliminate or reducer the adverse musculoskeletaleffects induced by treatment with an anti-hyaluronan agent, such asPEGylated hyaluronan degrading enzyme. In the methods, thecorticosteroid is used in a dosing regime or amount that does not affectthe therapeutic effect of the anti-hyaluronan agent, such as hyaluronandegrading enzyme. For example, anti-hyaluronan agents, such ashyaluronan degrading enzymes, and in particular polymer-conjugated suchas PEGylated hyaluronan degrading enzymes can be used for single therapyor combination therapy of a hyaluronan-associated disease or condition(see e.g. U.S. published application No. US2010003238 and Internationalpublished application No. WO2009128917). For example, a corticosteroidcan be used to ameliorate side effects or adverse events associated withtreatment of a hyaluronan-associated disease or condition, such as acancer, as described in Section H, and in particular single therapy orcombination therapy with a low dose PEGylated hyaluronan degradingenzyme (e.g. PEGPH20) that is found herein to exhibit therapeuticefficacy. Provided herein are pharmaceutical compositions, dosingschemes, and means of administration of corticosteroids to ameliorate orreduce the side effects of administered anti-hyaluronan agents. Thecompositions can be provided separately or together as part of a kit.

1. Pharmaceutical Compositions and Formulations

Pharmaceutical compositions containing a corticosteroid, particularlyglucocorticoids, such as dexamethasone, are provided herein.Corticosteroids, such as glucocorticoids, can be co-formulated orco-administered with pharmaceutical compositions containing ananti-hyaluronan agent, such as a PEGylated hyaluronan degrading enzymes,to ameliorate adverse side effects associated with treatment ofhyaluronan associated diseases or disorders with the anti-hyaluronanagent, e.g. PEGylated hyaluronidases.

Also provided herein are pharmaceutical compositions of anti-hyaluronanagents, for example, PEGylated hyaluronan degrading enzymes, such asPEGylated hyaluronidases. Also provided herein are pharmaceuticalcompositions containing a second agent that is used to treat a diseaseor disorder associated with a hyaluronan-associated disease orcondition, such as cancer. Exemplary of such agents include, but are notlimited to, anti-cancer agents including drugs, polypeptides, nucleicacids, antibodies, peptides, small molecules, gene therapy vector,viruses and other therapeutics. Anti-hyaluronan agents, such asPEGylated hyaluronan degrading enzymes, including PEGylatedhyaluronidases, such as PEGPH20, can be co-formulated or co-administeredwith pharmaceutical formulations of such second agents to enhance theirdelivery to desired sites or tissues within the body associated withexcess or accumulated hyaluronan.

Pharmaceutically acceptable compositions are prepared in view ofapprovals for a regulatory agency or other agency prepared in accordancewith generally recognized pharmacopeia for use in animals and in humans.The compounds can be formulated into any suitable pharmaceuticalpreparations for any of oral and intravenous administration such assolutions, suspensions, powders, or sustained release formulations.Typically, the compounds are formulated into pharmaceutical compositionsusing techniques and procedures well known in the art (see e.g., AnselIntroduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126.The formulation should suit the mode of administration.

In one example, pharmaceutical preparation can be in liquid form, forexample, solutions, syrups or suspensions. If provided in liquid form,the pharmaceutical preparations can be provided as a concentratedpreparation to be diluted to a therapeutically effective concentrationbefore use. Such liquid preparations can be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). In another example, pharmaceutical preparations can bepresented in lyophilized form for reconstitution with water or othersuitable vehicle before use.

Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which the composition (e.g.corticosteroid or anti-hyaluronan agent, such as a PEGylated hyaluronandegrading enzymes) are administered. Examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. Such compositions will contain a therapeutically effectiveamount of the compound or agent, generally in purified form or partiallypurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is atypical carrier. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Compositions can contain along with an activeingredient: a diluent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acacia, gelatin, glucose, molasses, polyvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, andethanol. For example, suitable excipients are, for example, water,saline, dextrose, glycerol or ethanol. A composition, if desired, alsocan contain other minor amounts of non-toxic auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, stabilizers,solubility enhancers, and other such agents, such as for example, sodiumacetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers, which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEENs 80). A sequestering or chelating agent of metalions include EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles andsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pHadjustment.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Preparationsfor intraprostatic administration include sterile solutions ready forinjection, sterile dry soluble products, such as lyophilized powders,ready to be combined with a solvent just prior to use, includinghypodermic tablets, sterile suspensions ready for injection, sterile dryinsoluble products ready to be combined with a vehicle just prior touse, sterile emulsions. The solutions can be either aqueous ornonaqueous.

2. Dosages and Administration

In the methods and uses provided herein, the corticosteroid isadministered in an amount to ameliorate, reduce or prevent a side effector adverse effect of an administered anti-hyaluronan agent. It isunderstood generally that it is the combination of the particularanti-hyaluronan agent and the route of administration that causes theparticular side effect. Exemplary of side effects observed by treatmentwith anti-hyaluronan agents are musculoskeletal side effects. The dosageor dosage regime at which an anti-hyaluronan agent causes an adverseside effect can be empirically determined by one of skill in the art asdescribed herein (see e.g. Section G). The Examples describe methods toassess and monitor adverse events in various subject and models asexemplified by administration of PEGPH20. Typically, the amount ofanti-hyaluronan agent also is an amount that achieves a therapeuticeffect, such as by inhibition of HA synthesis or degradation of HA. Theamount of corticosteroid that ameliorates, reduces or prevents theobserved side effects resulting from the administered anti-hyaluronanagent also can be empirically determined. The amount of corticosteroidis a function of the anti-hyaluronan agent, the route of administration,the observed or measured side effect and the particular patient orsubject.

Anti-hyaluronan agents and corticosteroids can be administered by anysuitable route of administration, including but not limited to orally,intravenously (IV), subcutaneously, intramuscularly, intra-tumorally,intradermally, topically, transdermally, rectally or sub-epidermally. Asdescribed herein, local administration also can be employed, inparticular for lower doses of an anti-hyaluronan agent. The route ofadministration of the anti-hyaluronan agent and corticosteroid can bethe same or different. The route of administration of theanti-hyaluronan agent is a function of the anti-hyaluronan agent and thedisease or condition to be treated. Likewise, the route ofadministration of the corticosteroid is a function of the particularcorticosteroid and the side effect which is being ameliorated.Typically, the route of administration of the corticosteroid andanti-hyaluronan agent is such so as to achieve a systemic effect. Forexample, PEGylated hyaluronan degrading enzymes are typicallyadministered intravenously. In another example, glucocorticoids aretypically administered orally. Other routes of administration arecontemplated, such as any route known to those of skill in the art.

The corticosteroid can be administered sequentially, intermittently, atthe same time or in the same composition as the anti-hyaluronan agent,such as the PEGylated hyaluronan degrading enzyme. Compositions also canbe administered with other biologically active second agents ortreatments, such as chemotherapeutic and biological agents, eithersequentially, intermittently, at the same time or in the samecomposition. Administration also can include controlled release systemsincluding controlled release formulations and device controlled release,such as by means of a pump.

Further, the concentration of the pharmaceutically active agent(s) isadjusted so that administration provides an effective amount to producethe desired pharmacological effect. The exact dose depends on the age,weight and condition of the patient or animal as is known in the art.The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. The volume of liquid solution orreconstituted powder preparation, containing the pharmaceutically activeagent(s), is a function of severity of the disease and the particulararticle of manufacture chosen for package. All preparations forparenteral administration must be sterile, as is known and practiced inthe art.

The pharmaceutical compositions can be formulated in dosage formsappropriate for each route of administration. Pharmaceuticallytherapeutically active agents and derivatives thereof are typicallyformulated and administered in unit dosage forms or multiple dosageforms. Each unit dose contains a predetermined quantity oftherapeutically active agent sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit dose forms can be administered in fractions or multiples thereof. Amultiple dose form is a plurality of identical unit dosage formspackaged in a single container to be administered in segregated unitdose form. Examples of multiple dose forms include vials, bottles oftablets or capsules or bottles of pints or gallons. Hence, a multipledose form is a multiple of unit doses that are not segregated inpackaging. Generally, dosage forms or compositions containing activeingredient in the range of 0.005% to 100% with the balance made up fromnon-toxic carrier can be prepared. The therapeutic agent(s) can beformulated as pharmaceutical compositions for single or multiple dosageuse.

a. Corticosteroid

A corticosteroid is administered is an amount that is therapeuticallyeffective to ameliorate or reduce the one or more adverse effects ofadministration of an anti-hyaluronan agent, such as a PEGylatedhyaluronan degrading enzymes, in particular, adverse musculoskeletaleffects. A therapeutically effective amount is the dosage sufficient toameliorate, prevent, eliminate or reduce one or more symptoms or adverseeffects. Indicators of improvement or successful pretreatment includedetermination of the failure to manifest a relevant score on the CTCAEscale or a change in grading or severity on the CTCAE scale.

The corticosteroid is provided in a therapeutically effective dose.Therapeutically effective concentration can be determined empirically bytesting in known in vitro or in vivo (e.g. animal model) systems. Forexample, the amount of a selected corticosteroid to be administered toameliorate the adverse effects can be determined by standard clinicaltechniques. In addition, animal models can be employed to help identifyoptimal dosage ranges. The precise dosage, which can be determinedempirically, can depend on the particular therapeutic preparation, theregime and dosing schedule, the route of administration and theseriousness of the disease. Methods of assessing such parameters aredescribed in Section G, and exemplified in Examples.

The concentration of a selected therapeutic agent in the compositiondepends on absorption, inactivation and excretion rates, thephysicochemical characteristics, the dosage schedule, and amountadministered as well as other factors known to those of skill in theart. For example, it is understood that the precise dosage and durationof treatment is a function of the disease or condition, the tissue beingtreated, the patient or subject and the anti-hyaluronan agent, includingamount and dosage regime. The dose of the corticosteroid also can varydepending on the age and health of the patient, the anti-hyaluronanagent dosing (e.g. PEGylated hyaluronan degrading enzyme dosing),potency of the corticosteroid, and the route of administration. Forexample, it is to be noted that concentrations and dosage values willvary with the therapeutic dose and dosage regime of the anti-hyaluronanagent, for example, the PEGylated hyaluronan degrading enzyme.Additionally, the corticosteroid can be administered daily, weekly, ormonthly or over longer periods of time in order to achieve the desiredresults. The particular dosage volume can vary and is dependent on thedosage regime, frequency of administration and the desired rate ofadministration. It is to be noted that concentrations and dosage valuescan also vary with the age of the individual treated.

The precise dosage and duration of treatment can be determinedempirically using known testing protocols or by extrapolation from invivo or in vitro test data. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope thereof.Generally, dosage regimens are chosen to limit toxicity, and herein arechosen to ameliorate adverse side effects. It should be noted that theattending physician would know how to and when to terminate, interruptor adjust therapy to lower dosage due to toxicity, or bone marrow, liveror kidney or other tissue dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects). Administration of a therapeutic agent should not exceed themaximum dosage levels established by the United States Food and DrugAdministration or published in the Physician's Desk Reference.

Generally, the dose of corticosteroid administered is dependent upon thespecific corticosteroid, as a difference in potency exists betweendifferent corticosteroids (see Table 4 below). The corticosteroid, orglucocorticoid, for example dexamethasone, can be given orally (tablets,liquid or liquid concentrate) PO, intravenously (IV) or intramuscularly.The corticosteroid is typically administered as a bolus, but many beadministered over a period of time, as long as the dose is effective toameliorate one or more side effects associated with administration ofthe anti-hyaluronan agent, for example, a PEGylated hyaluronidase.

TABLE 4 Glucocorticoid administration Glucocorticoid (route) EquivalentPotency (mg) Hydrocortisone (IV or PO) 20  Prednisone 5 Prednisolone (IVor PO) 5 Methylprednisolone sodium succinate (IV) 4 Dexamethasone (IV orPO) 0.5-0.75

The corticosteroid can be administered in any amount that is effectiveto ameliorate one or more side effects associated with administration ofthe anti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme. Thus, the corticosteroid, e.g., glucocorticoid, can beadministered, for example, at an amount between at or about 0.1 and 100mgs, per dose, 0.1 to 80 mgs, 0.1 to 60 mgs, 0.1 to 40 mgs, 0.1 to 30mgs, 0.1 to 20 mgs, 0.1 to 15 mgs, 0.1 to 10 mgs, 0.1 to 5 mgs, 0.2 to40 mgs, 0.2 to 30 mgs, 0.2 to 20 mgs, 0.2 to 15 mgs, 0.2 to 10 mgs, 0.2to 5 mgs, 0.4 to 40 mgs, 0.4 to 30 mgs, 0.4 to 20 mgs, 0.4 to 15 mgs,0.4 to 10 mgs, 0.4 to 5 mgs, 0.4 to 4 mgs, 1 to 20 mgs, 1 to 15 mgs or 1to 10 mgs, to a 70 kg adult human subject. Typically, thecorticosteroid, such as a glucocorticoid is administered at an amountbetween at or about 0.4 and 20 mgs, for example, at or about 0.4 mgs,0.5 mgs, 0.6 mgs, 0.7 mgs, 0.75 mgs, 0.8 mgs, 0.9 mgs, 1 mg, 2 mgs, 3mgs, 4 mgs, 5 mgs, 6 mgs, 7 mgs, 8 mgs, 9 mgs, 10 mgs, 11 mgs, 12 mgs,13 mgs, 14 mgs, 15 mgs, 16 mgs, 17 mgs, 18 mgs, 19 mgs or 20 mgs perdose, to an average adult human subject.

The corticosteroid can be administered, for example, at a dosage of ator about 0.001 mg/kg (of the subject), 0.002 mg/kg, 0.003 mg/kg, 0.004mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg,0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.03 mg/kg, 0.035mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg, 0.055 mg/kg, 0.06 mg/kg,0.065 mg/kg, 0.07 mg/kg, 0.075 mg/kg, 0.08 mg/kg, 0.085 mg/kg, 0.09mg/kg, 0.095 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.30mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80 mg/kg, 0.85 mg/kg, 0.90mg/kg, 0.95 mg/kg, 1 mg/kg, 1.05 mg/kg, 1.1 mg/kg, 1.15 mg/kg, 1.20mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg or 1.4 mg/kg, to an averageadult human subject, typically weighing about 70 kg to 75 kg.

The dosage administered administration can vary as long asadministration of the corticosteroid ameliorates one or more adverseside effects associated with administration of the anti-hyaluronanagent, such as a PEGylated hyaluronidase. In one example, the dosage ofglucocorticoid, for example, dexamethasone, is adminstered insuccessively lower dosages per treatment cycle. Hence, in such treatmentregimes, the dose of corticosteroid is tapered. For example,dexamethasone is administered prior to administration of ananti-hyaluronan agent, for example a PEGylated hyaluronidase, at aninitial dose of 4 mg, and upon each successive administration of theanti-hyaluronan agent, e.g. a PEGylated hyaluronidase, the dexamethasonedose is lowered, such that the dose is 3 mg for the next administrationof the anti-hyaluronan agent, e.g. PEGylated hyaluronidase, then 2 mgper administration of anti-hyaluronan agent, e.g. PEGylatedhyaluronidase, and then 1 mg per administration of anti-hyaluronanagent, e.g. PEGylated hyaluronidase. Any dose is contemplated as long asthe dose of the corticosteroid is effective to reduce one or more sideeffects associated with administration of the anti-hyaluronan agent,e.g. a PEGylated hyaluronidase.

Time of administration can vary as long as administration of thecorticosteroid ameliorates one or more adverse side effects associatedwith administration of the anti-hyaluronan agent, such as a PEGylatedhyaluronidase. The corticosteroid can be administered sequentially,intermittently, at the same time or in the same composition as theanti-hyaluronan agent, e.g. PEGylated hyaluronan degrading enzyme. Forexample, the corticosteroid can be administered before, during,simultaneously with, or after administration of the anti-hyaluronanagent, e.g. PEGylated hyaluronidase. In another example, thecorticosteroid and anti-hyaluronan agent, e.g. PEGylated hyaluronidaseare administered intermittently. Generally, the corticosteroid isadministered prior to administration of the anti-hyaluronan agent, e.g.PEGylated hyaluronidase. For example, the corticosteroid, e.g.,glucocorticoid, such as dexamethasone, can be administered at or about0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 18 hours, 24 hours, 36 hours or more prior to administrationof the anti-hyaluronan agent, for example a PEGylated hyaluronandegrading enzyme.

In some examples, the corticosteroid is administered at the same time asadministration of the anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzyme. In this example, the corticosteroid can beadministered together with, or separately from, the anti-hyaluronanagent, e.g. a PEGylated hyaluronidase. Typically, the corticosteroid isadministered separately from the anti-hyaluronan agent, for example aPEGylated hyaluronan degrading enzyme. In other examples, thecorticosteroid is administered at or about 0.5 minutes, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18hours, 24 hours, 36 hours or more after administration of theanti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme.

In one example, the corticosteroid is administered prior toadministration of anti-hyaluronan agent, for example a PEGylatedhyaluronidase. For example, the corticosteroid, e.g., glucocorticoid,for example, dexamethasone, is administered 1 hour prior to theadministration of the anti-hyaluronan agent, e.g. a PEGylatedhyaluronidase. In another example, the corticosteroid is administered 5minutes before the administration of the anti-hyaluronan agent, e.g. aPEGylated hyaluronan degrading enzyme. In another example, thecorticosteroid is administered both prior to and after theadministration of the anti-hyaluronan agent, e.g. a PEGylatedhyaluronidase. In this example, the corticosteroid, such asdexamethasone, is administered one to five minutes immediately beforeadministration of the anti-hyaluronan agent, e.g. a PEGylated hyaluronandegrading enzyme and eight hours after administration of theanti-hyaluronan agent, e.g. a PEGylated hyaluronan degrading enzyme. Inanother example, a corticosteroid, such as dexamethasone, isadministered one hour before administration of the anti-hyaluronanagent, e.g. a PEGylated hyaluronan degrading enzyme and eight to twelvehours after administration of the anti-hyaluronan agent, e.g. aPEGylated hyaluronan degrading enzyme.

Any dosing regime is contemplated as long as the time of dosing of thecorticosteroid ameliorates the one or more side effects associated withadministration of the anti-hyaluronan agent, for example a PEGylatedhyaluronidase. In addition, the dose or dosing regime of corticosteroidis one that does not interfere or reduce the therapeutic effect of theanti-hyaluronan agent in treating a hyaluronan associated disease ordisorder.

b. Anti-Hyaluronan Agent

In the method herein, the corticosteroid is administered to ameliorateor reduce adverse events associated with an anti-hyaluronan agent. Theamount of anti-hyaluronan agent that is administered is in an amountthat results in or causes an adverse side effect, such as amusculoskeletal side effect. Typically, the dose of hyaluronan-degradingenzyme is one that also achieves a therapeutic effect in the treatmentof a hyaluronan associated disease or condition. Hence, compositions ofan anti-hyaluronan agent are included in an amount sufficient to exert atherapeutically useful effect. The composition containing the activeagent can include a pharmaceutically acceptable carrier. Thecompositions of an anti-hyaluronan agent also can include a secondagent. Generally, the dosing (amount and dosing regime) of theanti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme, will determine the dosing (amount and dosing regime) of thecorticosteroid, e.g. a glucocorticoid.

Therapeutically effective concentration of anti-hyaluronan agents can bedetermined empirically by testing the compounds in known in vitro and invivo systems, such as the assays provided herein. For example, theconcentration of an anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzyme, such as PEGylated hyaluronidase depends onabsorption, inactivation and excretion rates of the complex, thephysicochemical characteristics of the complex, the dosage schedule, andamount administered as well as other factors known to those of skill inthe art. For example, it is understood that the precise dosage andduration of treatment is a function of the tissue being treated, thedisease or condition being treated, the route of administration, thepatient or subject and the particular anti-hyaluronan agent and can bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data and/or can be determined from knowndosing regimes of the particular agent. The amount of an anti-hyaluronanagent, for example a PEGylated hyaluronan degrading enzyme, such as aPEGylated hyaluronidase, to be administered for the treatment of adisease or condition, for example a hyaluronan-associated disease orcondition such as an HA-rich tumor, can be determined by standardclinical techniques. In addition, in vitro assays and animal models canbe employed to help identify optimal dosage ranges. The precise dosage,which can be determined empirically, can depend on the particularenzyme, the route of administration, the type of disease to be treatedand the seriousness of the disease.

For example, agents and treatments for treatment ofhyaluronan-associated diseases and conditions, such as anti-canceragents, are well known in the art (see e.g. U.S. published applicationNo. 20100003238 and International published PCT Appl. No. WO2009/128917). Thus, dosages of an anti-hyaluronan agent, such as ahyaluronan-degrading enzyme for example a hyaluronidase, or other secondagents in a composition can be chosen based on standard dosing regimesfor that agent under a given route of administration. Also, as describedin Section H below, it is found herein that far lower doses of ahyaluronan degrading enzyme, for example, exhibit therapeutic efficiacyfor the treatment of hyaluronan-associated disease and condition. Hence,dosage regimens using lower doses of a hyaluronan degrading enzyme, forexample a polymer-modified hyaluronan-degrading enzyme such as aPEGylated PH20, can be used to treat a hyaluronan-associated disease andcondition. As described herein, any of such dosage or dosage regimen canbe associated with adverse events, such as musculoskeletal side effects,which can be ameliorated by premedication, co-administration and/orpost-medication of a corticosteroid.

Examples of effective amounts of an anti-hyaluronan agent is a doseranging from 0.01 μg to 100 g per kg of body weight. For example, aneffective amount of an anti-hyaluronan agent is a dose ranging from 0.01μg to 100 mg per kg of body weight, such as 0.01 μg to 1 mg per kg ofbody weight, 1 μg to 100 μg per kg of body weight, 1 μg to 10 μg per kgof body weight or 0.01 mg to 100 mg per kg of body weight. For example,effective amounts include at least or about at least or about or 0.01μg, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900 or 1000 μg/kg body weight., Other examples ofeffective amounts include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 g/kg body weight. For example, an anti-hyaluronanagent, such as a hyaluronan-degrading enzyme for example a hyaluronidase(e.g. a PEGylated hyaluronidase such as a PEGPH20), can be administeredat or about 0.1 μg/kg to 1 mg/kg, for example 0.5 μg/kg to 100 μg/kg,0.75 μg/kg to 15 μg/kg, 0.75 μg/kg to 7.5 μg/kg or 1.0 μg/kg to 3.0μg/kg. In other examples, an anti-hyaluronan agent such as ahyaluronan-degrading enzyme for example a hyaluornidase (e.g. aPEGylated hyaluronidase such as a PEGPH20), can be administered at or 1mg/kg to 500 mg/kg, for example, 100 mg/kg to 400 mg/kg, such as 200mg/kg. Generally, compositions contain 0.5 mg to 100 grams ofanti-hyaluronan agent, for example, 20 μg to 1 mg, such as 100 μg to 0.5mg or can contain 1 mg to 1 gram, such as 5 mg to 500 mg.

The dose or compositions can be for single dosage administration or formultiple dosage administration. The dose or composition can beadministered in a single administration once, several times a week,twice weekly, every 15 days, 16 days, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 days, once monthly, several times a year oryearly. In other examples, the dose or composition an be divided up andadministered once, several times a week, twice weekly, every 15 days, 16days, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days,once monthly, several times a year or yearly. Anti-hyaluronancompositions can be formulated as liquid compositions or can belyophilized. The compositions also can be formulated as a tablet orcapsule.

Provided below is description of dosages and dosage regimines ofexemplary anti-hyaluronan agents for use in the methods herein. Theanti-hyaluronan agents can be used alone in a single agent therapy or incombination with other agents for use in treating an HA-associateddisease or condition. As discussed elsewhere herein, in particularexamples of the methods and uses herein, that agents are administered incombination with a corticosteroid in order to ameliorate a side-effectassociated with treatment of the anti-hyaluronan-agent.

i. Leflunomide and Derivatives

In one example, leflunomide, or derivatives thereof, generally areavailable as tablets containing 1-100 mg of active drug, for example, 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg of drug. For thetreatment of hyaluronan associated diseases and conditions, for exampleRheumatoid arthritis, it is administered at 10 to 500 mg per day,typically 100 mg per day. The dosage can be continued as needed fortreatment of the disease or conditions, or can be tapered or reduced tosuccessively lower doses. For example, for treatment of Rheumatoidarthritis, leflunomide can be administered at an initial loading dose of100 mg per day for three days and then administered at a continued doseof 20 mg/day.

ii. Hyaluronan-Degrading Enzyme

For example, a hyaluronan-degrading enzyme, such as a PEGylatedhyaluronan degrading enzyme (e.g. a hyaluronidase), can be administeredsystemically, for example, intravenously (IV), intramuscularly, or byany another systemic route. In particular examples, lower doses can begiven locally. For example, local administration of ahyaluronan-degrading enzyme, such as a PEGylated hyaluronan degradingenzyme for example a PEGylated hyaluronidase (e.g. PH20) includesintratumoral administration, arterial injection (e.g. hepatic artery),intraperitoneal administration, intravesical administration and otherlocal routes used for cancer therapy that can increase local action at alower absolute dose.

Exemplary dosage range is at or about 0.3 Units/kg to 320,000 Units/kg,such as 10 Units/kg to 320,000 Units/kg of a PEGylated hyaluronidase, ora functionally equivalent amount of another PEGylated hyaluronandegrading enzyme. It is understood herein that a unit of activity isnormalized to a standard activity, for example, an activity as measuredin a microturbidity assay assaying hyaluronidase activity. A PEGylatedsoluble hyaluronidase can exhibit lower activity per mg of totalprotein, i.e. exhibits a lower specific activity, compared to a nativesoluble hyaluronidase not so conjugated. For example, an exemplaryrHuPH20 preparation exhibits a specific activity of 120,000 Units/mg,while a PEGylated form of rHuPH20 exhibits a specific activity of at orabout 32,000 Units/mg. Typically, a PEGylated form of ahyaluronan-degrading enzyme, such as a hyaluronidase for examplerHuPH20, exhibits a specific activity within the range of between at orabout 18,000 and at or about 45,000 U/mg. In one example, thePEG-hyaluronan degrading enzyme can be provided as a stock solution forexample, at 3.5 mg/mL at 112,000 U/mL (˜32,000 U/mg), with a PEG toprotein molar ratio between 5:1 and 9:1, for example, 7:1, or can beprovided in a less concentrated form. For purposes herein, dosages canbe with reference to Units.

For example, PEGylated hyaluronan degrading enzyme, such as ahyaluronidase, for example PEGPH20, can be administered intravenouslytwice weekly, once weekly or once every 21 days. Typically, thePEGylated hyaluronan degrading enzyme is administered twice weekly. Thecycle of administration can be for a defined period, generally for 3weeks or 4 weeks. The cycle of administration can be repeated in adosage regime for more than one month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1year or more. Generally, the cycle of administration is repeated at thediscretion of a treating physician, and can depend on factors such asremission of the disease or condition, severity of the disease orcondition, adverse events and other factors. In other examples, insubsequent cycles of administration, the hyaluronan-degrading enzyme canbe administered less frequently. For example, in a first cycle thehyaluronan-degrading enzyme is administered twice weekly for four weeks,and in subsequent cycles of administration the hyaluronan-degradingenzyme is administered once weekly or once every two weeks, once every 3weeks (e.g. once every 21 days) or once every 4 weeks. As describedherein, the dose or dosing regime of corticosteroid is dependent on thedosing regime of hyaluronan-degrading enzyme.

While dosages can vary depending on the disease and patient, thehyaluronan-degrading enzyme, such as a PEGylated hyaluronidase, isgenerally administered in an amount that is or is about in the range offrom 0.01 μg/kg to 25 mg/kg, such as 0.0005 mg/kg (0.5 μg/kg) to 10mg/kg (320,000 U/kg), for example, 0.02 mg/kg to 1.5 mg/kg, for example,0.05 mg/kg. The PEGylated hyaluronidase can be administered, forexample, at a dosage of at or about 0.0005 mg/kg (of the subject),0.0006 mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg,0.0016 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.016 mg/kg,0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg,0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg,0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg kg,0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg,1.9 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, or more is administered,to an average adult human subject, typically weighing about 70 kg to 75kg. In particular examples, as described in Section H herein, thehyaluronan-degrading enzyme is administered in lower amounts such asless than 20 μg/kg, for example 0.01 μg/kg to 15 μg/kg, 0.05 μg/kg to 10μg/kg, 0.75 μg/kg to 7.5 μg/kg or 1.0 μg/kg to 3.0 μg/kg.

A hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase (e.g.PH20), provided herein, for example, PEGPH20, can be administered at orabout 1 Unit/kg to 800,000 Units/kg, such as 10 to 800,000 Units/kg, 10to 750,000 Units/kg, 10 to 700,000 Units/kg, 10 to 650,000 Units/kg, 10to 600,000 Units/kg, 10 to 550,000 Units/kg, 10 to 500,000 Units/kg, 10to 450,000 Units/kg, 10 to 400,000 Units/kg, 10 to 350,000 Units/kg, 10to 320,000 Units/kg, 10 to 300,000 Units/kg, 10 to 280,000 Units/kg, 10to 260,000 Units/kg, 10 to 240,000 Units/kg, 10 to 220,000 Units/kg, 10to 200,000 Units/kg, 10 to 180,000 Units/kg, 10 to 160,000 Units/kg, 10to 140,000 Units/kg, 10 to 120,000 Units/kg, 10 to 100,000 Units/kg, 10to 80,000 Units/kg, 10 to 70,000 Units/kg, 10 to 60,000 Units/kg, 10 to50,000 Units/kg, 10 to 40,000 Units/kg, 10 to 30,000 Units/kg, 10 to20,000 Units/kg, 10 to 15,000 Units/kg, 10 to 12,800 Units/kg, 10 to10,000 Units/kg, 10 to 9,000 Units/kg, 10 to 8,000 Units/kg, 10 to 7,000Units/kg, 10 to 6,000 Units/kg, 10 to 5,000 Units/kg, 10 to 4,000Units/kg, 10 to 3,000 Units/kg, 10 to 2,000 Units/kg, 10 to 1,000Units/kg, 10 to 900 Units/kg, 10 to 800 Units/kg, 10 to 700 Units/kg, 10to 500 Units/kg, 10 to 400 Units/kg, 10 to 300 Units/kg, 10 to 200Units/kg, 10 to 100 Units/kg, 16 to 600,000 Units/kg, 16 to 500,000Units/kg, 16 to 400,000 Units/kg, 16 to 350,000 Units/kg, 16 to 320,000Units/kg, 16 to 160,000 Units/kg, 16 to 80,000 Units/kg, 16 to 40,000Units/kg, 16 to 20,000 Units/kg, 16 to 16,000 Units/kg, 16 to 12,800Units/kg, 16 to 10,000 Units/kg, 16 to 5,000 Units/kg, 16 to 4,000Units/kg, 16 to 3,000 Units/kg, 16 to 2,000 Units/kg, 16 to 1,000Units/kg, 16 to 900 Units/kg, 16 to 800 Units/kg, 16 to 700 Units/kg, 16to 500 Units/kg, 16 to 400 Units/kg, 16 to 300 Units/kg, 16 to 200Units/kg, 16 to 100 Units/kg, 160 to 12,800 Units/kg, 160 to 8,000Units/kg, 160 to 6,000 Units/kg, 160 to 4,000 Units/kg, 160 to 2,000Units/kg, 160 to 1,000 Units/kg, 160 to 500 Units/kg, 500 to 5000Units/kg, 1000 to 100,000 Units/kg or 1000 to 10,000 Units/kg, of themass of the subject to whom it is administered. In some examples, ahyaluronan-degrading enzyme, such as a PEGylated hyaluronidase (e.g.PH20), provided herein, for example, PEGPH20, can be administered at orabout 1 Unit/kg to 1000 Units/kg, 1 Units/kg to 500 Units/kg or 10Units/kg to 50 Units/kg.

Generally, where the specific activity of the PEGylated hyaluronidase isor is about 18,000 U/mg to 45,000 U/mg, generally at or about 1 Units/kg(U/kg), 2 U/kg, 3 U/kg, 4 U/kg, 5 U/kg, 6 U/kg, 7 U/kg, 8 U/kg, 8 U/kg10 U/kg, 16 U/kg, 32 U/kg, 64 U/kg, 100 U/kg, 200 U/kg, 300 U/kg, 400U/kg, 500 U/kg, 600 U/kg, 700 U/kg, 800 U/kg, 900 U/kg, 1,000 U/kg,2,000 U/kg, 3,000 U/kg, 4,000 U/kg, 5,000 U/kg, 6,000 U/kg, 7,000 U/kg,8,000 U/kg, 9,000 U/kg, 10,000 U/kg, 12,800 U/kg, 20,000 U/kg, 32,000U/kg, 40,000 U/kg, 50,000 U/kg, 60,000 U/kg, 70,000 U/kg, 80,000 U/kg,90,000 U/kg, 100,000 U/kg, 120,000 U/kg, 140,000 U/kg, 160,000 U/kg,180,000 U/kg, 200,000 U/kg, 220,000 U/kg, 240,000 U/kg, 260,000 U/kg,280,000 U/kg, 300,000 U/kg, 320,000 U/kg, 350,000 U/kg, 400,000 U/kg,450,000 U/kg, 500,000 U/kg, 550,000 U/kg, 600,000 U/kg, 650,000 U/kg,700,000 U/kg, 750,000 U/kg, 800,000 U/kg or more, per mass of thesubject, is administered.

In some aspects, the PEGylated hyaluronan degrading enzyme is formulatedand dosed to maintain at least 3 U/mL of the PEGylated hyaluronidase inthe plasma (see e.g. published U.S. Patent App. No. US20100003238 andpublished International Patent App. No. WO2009128917). For example, thePEGylated soluble hyaluronidase is formulated for systemicadministration in a sufficient amount to maintain at least or about 3U/mL in the plasma, generally 3 U/mL-12 U/mL or more, for example, fromat least or about or at a level of 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8U/mL, 9 U/mL, 10 U/mL, 11 U/mL, 12 U/mL, 13 U/mL, 14 U/mL, 15 U/mL, 16U/mL, 17 U/mL, 18 U/mL, 19 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 40U/mL, 45 U/mL, 50 U/mL or more. Generally, for purposes herein tomaintain at least 3 U/mL of the hyaluronidase in plasma, at or about0.02 mg/kg (of the subject), 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg or more isadministered. Generally, where the specific activity of the modifiedhyaluronidase is or is about 20,000 U/mg to 60,000 U/mg, generally at orabout 35,000 U/mg, 60,000 U; 70,000 U; 80,000 U; 90,000 U; 100,000 U;200,000 U; 300,000 U; 400,000 U; 500,000 U; 600,000 U; 700,000 U;800,000 U; 900,000 U; 1,000,000 U; 1,500,000 U; 2,000,000 U; 2,500,000U; 3,000,000 U; 3,500,000 U; 4,000,000 U or more is administered. Tomaintain such levels, administration can be daily, several times a week,twice weekly, weekly or monthly.

It is within the level of one of skill in the art to determine theamounts of PEGylated hyaluron degrading enzyme, for example, PEGylatedPH20, to maintain at least 3 U/mL of the hyaluronidase in the blood. Thelevel of hyaluronidase in the blood can be monitored over time in orderto ensure that a sufficient amount of the hyaluronidase is present inthe blood. Any assay known to one of skill in the art to measure thehyaluronidase in the plasma can be performed. For example, amicroturbidity assay or enzymatic assay described in the Examples hereincan be performed on protein in plasma. It is understood that plasmanormally contains hyaluronidase enzymes. Such plasma hyaluronidaseenzymes typically have activity at an acidic pH (U.S. Pat. No.7,105,330). Hence, before treatment of with a modified enzyme, theplasma levels of hyaluronidase should be determined and used as abaseline. Subsequent measurements of plasma hyaluronidase levels aftertreatment can be compared to the levels before treatments.Alternatively, the assay can be performed under pH conditions thatsuppress endogenous lysosomal hyaluronidase activity in plasma, whichnormally exhibits activity at acidic pH. Thus, where the modifiedsoluble hyaluronidase is active at neutral pH (e.g. human PH20), onlythe level of the modified neutral-active soluble hyaluronidase ismeasured.

In other examples, as described in Section H herein, the PEGylatedhyaluronan degrading enzyme is formulated and administered at a lowerdose, which is found herein to have therapeutic effects to treat ahyaluronan-associated disease or conditions absent a detectable level ofhyaluronidase maintained in the blood. For example, the PEGylatedsoluble hyaluronidase is administered in an amount that is less than 20μg/kg, for example 0.01 μg/kg to 15 μg/kg, 0.05 μg/kg to 10 μg/kg, 0.75μg/kg to 7.5 μg/kg or 1.0 μg/kg to 3.0 μg/kg, such as at or about 0.01μg/kg (of the subject), 0.02 μg/kg, 0.03 μg/kg, 0.04 μg/kg, 0.05 μg/kg,1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 2.5 μg/kg, 3.0 μg/kg, 3.5 μg/kg, 4.0μg/kg, 4.5 μg/kg, 5.0 μg/kg, 5.5 μg/kg, 6.0 μg/kg, 7.0 μg/kg, 7.5 μg/kg,8.0 μg/kg, 9.0 μg/kg, 10.0 μg/kg, 12.5 μg/kg or 15 μg/kg. Generally,where the specific activity of the modified hyaluronidase is or is about20,000 U/mg to 60,000 U/mg, generally at or about 35,000 U/mg, 200 Unitsto 50,000 (U) is administered, such as 200 U, 300 U; 400 U; 500 U; 600U; 700 U; 800 U; 900 U; 1,000 U; 1250 U; 1500 U; 2000 U; 3000 U; 4000 U;5,000 U; 6,000 U; 7,000 U; 8,000 U; 9,000 U; 10,000 U; 20,000 U; 30,000U; 40,000 U; or 50,000 U is administered. To maintain such levels,administration can be daily, several times a week, twice weekly, weeklyor monthly.

Typically, volumes of injections or infusions of PEGylated hyaluronidasecontemplated herein are from at or about 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL,5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50 mLor more. The PEGylated hyaluronan degrading enzyme, such as a PEGylatedhyaluronidase can be provided as a stock solution at or about 50 U/mL,100 U/mL, 150 U/mL, 200 U/mL, 400 U/mL or 500 U/mL (or a functionallyequivalent amount) or can be provided in a more concentrated form, forexample at or about 1000 U/mL, 2000 Units/mL, 3000 U/mL, 4000 U/mL, 5000U/mL, 6000 U/mL, 7000 U/mL, 8000 U/mL, 9000 U/mL, 10,000 U/mL, 11,000U/mL, 12,000 U/mL, or 12,800 U/mL, for use directly or for dilution tothe effective concentration prior to use. The volume of PEGylatedhyaluronan degrading enzyme, such as PEGylated hyaluronidase,administered is a function of the dosage required, but can be varieddepending on the concentration of a hyaluronan degrading enzyme, such assoluble hyaluronidase, stock formulation available. For example, it iscontemplated herein that the PEGylated hyaluronan degrading enzyme, suchas PEGylated hyaluronidase, is not administered in volumes greater thanabout 50 mL, and typically is administered in a volume of 5-30 mL,generally in a volume that is not greater then about 10 mL. ThePEGylated hyaluronan degrading enzyme, such as a PEGylatedhyaluronidase, can be provided as a liquid or lyophilized formulation.Lyophilized formulations are ideal for storage of large unit doses ofPEGylated hyaluronan degrading enzymes.

3. Combination Treatment

Anti-hyaluronan agents, such as a hyaluronan-degrading enzymes forexample a PEGylated hyaluronan degrading enzyme (e.g. PEGylatedhyaluronaidase such as PEGPH20) can be administered in a combinationtreatment, for example, for the treatment of a hyaluronan-associateddisease or condition. As described herein, a corticosteroid can beadministered to ameliorate side effects or adverse events of theanti-hyaluronan agent in the combination therapy.

For example, compositions of an anti-hyaluronan agent described hereincan be further co-formulated or co-administered together with, prior to,intermittently with, or subsequent to, other therapeutic orpharmacologic agents or treatments, such as procedures, for example,agents or treatments to treat a hyaluronan associated disease orcondition, for example hyaluronan-associated cancers. Such agentsinclude, but are not limited to, other biologics, anti-cancer agents,small molecule compounds, dispersing agents, anesthetics,vasoconstrictors and surgery, and combinations thereof. Such otheragents and treatments that are available for the treatment of a diseaseor condition, including all those exemplified herein, are known to oneof skill in the art or can be empirically determined.

A preparation of a second agent or agents or treatment or treatments canbe administered at once, or can be divided into a number of smallerdoses to be administered at intervals of time. Selected agent/treatmentpreparations can be administered in one or more doses over the course ofa treatment time for example over several hours, days, weeks, or months.In some cases, continuous administration is useful. It is understoodthat the precise dosage and course of administration depends on theindication and patient's tolerability. Generally, dosing regimes forsecond agents/treatments herein are known to one of skill in the art.

In one example, an anti-hyaluronan agent, such as a PEGylated hyaluronandegrading enzyme, such as PEGylated hyaluronidase, is administered aspart of a combination therapy, by administering the anti-hyaluronanagent (e.g. a PEGylated hyaluronan degrading enzyme) and a second agentor treatment for treating the disease or condition. In one example, theanti-hyaluronan agent, such as a PEGylated hyaluronan degrading enzyme,and second agent or treatment can be co-formulated and administeredtogether. In another example, the anti-hyaluronan agent, such as aPEGylated hyaluronan degrading enzyme, such as PEGylated hyaluronidase,is administered subsequently, intermittently or simultaneously with thesecond agent or treatment preparation. Generally, the anti-hyaluronanagent, for example a PEGylated hyaluronan degrading enzyme, isadministered prior to administration of the second agent or treatmentpreparation to permit the anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzyme, to reduce or degrade the hyaluronic acid ina cell, tissue or fluid of the subject, such as, for example, theinterstitial space, extracellular matrix, tumor tissue, blood or othertissue. For example, an anti-hyaluronan agent, such as a PEGylatedhyaluronan degrading enzyme, such as soluble hyaluronidase, can beadministered 0.5 minutes, 1 minute, 2 minute, 3 minute, 4 minute, 5minute, 6 minute, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20minutes, 30 minutes, 1 hour or more prior to administration of thesecond agent preparation. In some examples, the anti-hyaluronan agent,for example a PEGylated hyaluronan degrading enzyme, is administeredtogether with the second agent preparation. As will be appreciated bythose of skill in the art, the desired proximity of co-administrationdepends in significant part in the effect half lives of the agents inthe particular tissue setting, and the particular disease being treated,and can be readily optimized by testing the effects of administering theagents at varying times in suitable models, such as in suitable animalmodels. In some situations, the optimal timing of administration of theanti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme, such as a PEGylated hyaluronidase, will exceed 60 minutes.

Anti-Cancer Agents and Other Treatments

The anticancer agent(s) or treatment(s) can be surgery, radiation,drugs, chemotherapeutics, polypeptides, antibodies, peptides, smallmolecules or gene therapy vectors, viruses or DNA.

Exemplary anti-cancer agents that can be administered after, coincidentwith or before administration of the PEGylated hyaluronan degradingenzyme, such as a PEGylated hyaluronidase, include, but are not limitedto Acivicins; Avicin; Aclarubicins; Acodazoles; Acronines; Adozelesins;Aldesleukins; Alemtuzumabs; Alitretinoins (9-Cis-Retinoic Acids);Allopurinols; Altretamines; Alvocidibs; Ambazones; Ambomycins;Ametantrones; Amifostines; Aminoglutethimides; Amsacrines; Anastrozoles;Anaxirones; Ancitabines; Anthramycins; Apaziquones; Argimesnas; ArsenicTrioxides; Asparaginases; Asperlins; Atrimustines; Azacitidines;Azetepas; Azotomycins; Banoxantrones; Batabulins; Batimastats; BCG Live;Benaxibines; Bendamustines; Benzodepas; Bexarotenes; Bevacizumab;Bicalutamides; Bietaserpines; Biricodars; Bisantrenes; Bisantrenes;Bisnafide Dimesylates; Bizelesins; Bleomycins; Bortezomibs; Brequinars;Bropirimines; Budotitanes; Busulfans; Cactinomycins; Calusterones;Canertinibs; Capecitabines; Caracemides; Carbetimers; Carboplatins;Carboquones; Carmofurs; Carmustines with Polifeprosans; Carmustines;Carubicins; Carzelesins; Cedefingols; Celecoxibs; Cemadotins;Chlorambucils; Cioteronels; Cirolemycins; Cisplatins; Cladribines;Clanfenurs; Clofarabines; Crisnatols; Cyclophosphamides; Cytarabineliposomals; Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;Decitabines; Denileukin Diftitoxes; Dexniguldipines; Dexonnaplatins;Dexrazoxanes; Dezaguanines; Diaziquones; Dibrospidiums; Dienogests;Dinalins; Disermolides; Docetaxels; Dofequidars; Doxifluridines;Doxorubicin liposomals; Doxorubicin HCL; Doxorubicin HCL liposomeinjection; Doxorubicins; Droloxifenes; Dromostanolone Propionates;Duazomycins; Ecomustines; Edatrexates; Edotecarins; Eflornithines;Elacridars; Elinafides; Elliott's B Solutions; Elsamitrucins; Emitefurs;Enloplatins; Enpromates; Enzastaurins; Epipropidines; Epirubicins;Epoetin alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;Etoposides; Etoprines; Exemestanes; ExisulindS; Fadrozoles; Fazarabines;Fenretinides; Filgrastims; Floxuridines; Fludarabines; Fluorouracils;5-fluorouracils; Fluoxymesterones; Fluorocitabines; Fosquidones;Fostriecins; Fostriecins; Fotretamines; Fulvestrants; Galarubicins;Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins; Geroquinols;Gimatecans; Gimeracils; Gloxazones; Glufosfamides; Goserelin acetates;Hydroxyureas; Ibritumomabs/Tiuxetans; Idarubicins; Ifosfamides;Ilmofosines; Ilomastats; Imatinib mesylates; Imexons; Improsulfans;Indisulams; Inproquones; Interferon alfa-2 as; Interferon alfa-2bs;Interferon Alfas; Interferon Betas; Interferon Gammas; Interferons;Interleukin-2s and other Interleukins (including recombinantInterleukins); Intoplicines; Iobenguanes [131-I]; Iproplatins;Irinotecans; Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines;Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins;Leuprolides; Leuprorelins (Leuprorelides); Levamisoles; Lexacalcitols;Liarozoles; Lobaplatins; Lometrexols; Lomustines/CCNUs; Lomustines;Lonafarnibs; Losoxantrones; Lurtotecans; Mafosfamides; Mannosulfans;Marimastats; Masoprocols; Maytansines; Mechlorethamines;Mechlorethamines/Nitrogen mustards; Megestrol acetates; Megestrols;Melengestrols; Melphalans; MelphalanslL-PAMs; Menogarils; Mepitiostanes;Mercaptopurines; 6-Mercaptopurine; Mesnas; Metesinds; Methotrexates;Methoxsalens; Metomidates; Metoprines; Meturedepas; Miboplatins;Miproxifenes; Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone Phenpropionates;Nedaplatins; Nelzarabines; Nemorubicins; Nitracrines; Nocodazoles;Nofetumomabs; Nogalamycins; Nolatrexeds; Nortopixantrones; Octreotides;Oprelvekins; Ormaplatins; Ortataxels; Oteracils; Oxaliplatins;Oxisurans; Oxophenarsines; Paclitaxels; Pamidronates; Patubilones;Pegademases; Pegaspargases; Pegfilgrastims; Peldesines; Peliomycins;Pelitrexols; Pemetrexeds; Pentamustines; Pentostatins; Peplomycins;Perfosfamides; Perifosines; Picoplatins; Pinafides; Pipobromans;Piposulfans; Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds;Plicamycid Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimersodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins; Sabarubicins;Safingols; Sargramostims; Satraplatins; Sebriplatins; Semustines;Simtrazenes; Sizofurans; Sobuzoxanes; Sorafenibs; Sparfosates; SparfosicAcids; Sparsomycins; Spirogermaniums; Spiromustines; Spiroplatins;Spiroplatins; Squalamines; Streptonigrins; Streptovarycins;Streptozocins; Sufosfamides; Sulofenurs; Sunitinib Malate; 6-thioguanine(6-TG); Tacedinalines; Talcs; Talisomycins; Tallimustines; Tamoxifens;Tariquidars; Tauromustines; Tecogalans; Tegafurs; Teloxantrones;Temoporfins; Temozolomides; Teniposides/VM-26s; Teniposides;Teroxirones; Testolactones; Thiamiprines; Thioguanines; Thiotepas;Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;Tirapazamines; Topixantrones; Topotecans; Toremifenes; Tositumomabs;Trabectedins (Ecteinascidin 743); Trastuzumabs; Trestolones;Tretinoins/ATRA; Triciribines; Trilostanes; Trimetrexates; TriplatinTetranitrates; Triptorelins; Trofosfamides; Tubulozoles; Ubenimexs;Uracil Mustards; Uredepas; Valrubicins; Valspodars; Vapreotides;Verteporfins; Vinblastines; Vincristines; Vindesines; Vinepidines;Vinflunines; Vinformides; Vinglycinates; Vinleucinols; Vinleurosines;Vinorelbines; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles;Xanthomycin As (Guamecyclines); Zeniplatins; Zilascorbs [2-H];Zinostatins; Zoledronate; Zorubicins; and Zosuquidars, for example:

Aldesleukins (e.g. PROLEUKIN®); Alemtuzumabs (e.g. CAMPATH®);Alitretinoins (e.g. PANRETIN®); Allopurinols (e.g. ZYLOPRIM®);Altretamines (e.g. HEXALEN®); Amifostines (e.g. ETHYOL®); Anastrozoles(e.g. ARIMIDEX®); Arsenic Trioxides (e.g. TRISENOX®); Asparaginases(e.g. ELSPAR®); BCG Live (e.g. TICE® BCG); Bexarotenes (e.g.TARGRETIN®); Bevacizumab (AVASTIN®); Bleomycins (e.g. BLENOXANE®);Busulfan intravenous (e.g. BUSULFEX®); Busulfan orals (e.g. MYLERAN®);Calusterones (e.g. METHOSARB®); Capecitabines (e.g. XELODA®);Carboplatins (e.g. PARAPLATIN®); Carmustines (e.g. BCNU®, BiCNU®);Carmustines with Polifeprosans (e.g. GLIADEL® Wafer); Celecoxibs (e.g.CELEBREX®); Chlorambucils (e.g. LEUKERAN®); Cisplatins (e.g. PLATINOL®);Cladribines (e.g. LEUSTATIN®, 2-CdA®); Cyclophosphamides (e.g. CYTOXAN®,NEOSAR®); Cytarabines (e.g. CYTOSAR-U®); Cytarabine liposomals (e.g.DepoCyt®); Dacarbazines (e.g. DTIC-Dome): Dactinomycins (e.g.COSMEGEN®); Darbepoetin Alfas (e.g. ARANESP®); Daunorubicin liposomals(e.g. DANUOXOME®); Daunorubicins/Daunomycins (e.g. CERUBIDINE®);Denileukin Diftitoxes (e.g. ONTAK®); Dexrazoxanes (e.g. ZINECARD®);Docetaxels (e.g. TAXOTERE®); Doxorubicins (e.g. ADRIAMYCIN®, RUBEX®);Doxorubicin liposomals, including Doxorubicin HCL liposome injections(e.g. DOXIL®); Dromostanolone propionates (e.g. DROMOSTANOLONE® andMASTERONE® Injection); Elliott's B Solutions (e.g. Elliott's BSolution®); Epirubicins (e.g. ELLENCE®); Epoetin alfas (e.g. EPOGEN®);Estramustines (e.g. EMCYT®); Etoposide phosphates (e.g. ETOPOPHOS®);Etoposide VP-16s (e.g. VEPESID®); Exemestanes (e.g. AROMASIN®);Filgrastims (e.g. NEUPOGEN®); Floxuridines (e.g. FUDR®); Fludarabines(e.g. FLUDARA®); Fluorouracils incl. 5-FUs (e.g. ADRUCIL®); Fulvestrants(e.g. FASLODEX®); Gemcitabines (e.g. GEMZAR®); Gemtuzumabs/Ozogamicins(e.g. MYLOTARG®); Goserelin acetates (e.g. ZOLADEX®); Hydroxyureas (e.g.HYDREA®); Ibritumomabs/Tiuxetans (e.g. ZEVALIN®); Idarubicins (e.g.IDAMYCIN®); Ifosfamides (e.g. IFEX®); Imatinib mesylates (e.g.GLEEVEC®); Interferon alfa-2 as (e.g. ROFERON-A®); Interferon alfa-2bs(e.g. INTRON A®); Irinotecans (e.g. CAMPTOSAR®); Letrozoles (e.g.FEMARA®); Leucovorins (e.g. WELLCOVORIN®, LEUCOVORIN®); Levamisoles(e.g. ERGAMISOL®); Lomustines/CCNUs (e.g. CeeBU®);Mechlorethamines/Nitrogen mustards (e.g. MUSTARGEN®); Megestrol acetates(e.g. MEGACE®); Melphalans/L-PAMs (e.g. ALKERAN®); Mercaptopurine,including 6-mercaptopurines (6-MPs; e.g. PURINETHOL®); Mesnas (e.g.MESNEX®); Methotrexates; Methoxsalens (e.g. UVADEX®); Mitomycin Cs (e.g.MUTAMYCIN®, MITOZYTREX®); Mitotanes (e.g. LYSODREN®); Mitoxantrones(e.g. NOVANTRONE®); Nandrolone Phenpropionates (e.g. DURABOLIN-50®);Nofetumomabs (e.g. VERLUMA®); Oprelvekins (e.g. NEUMEGA®); Oxaliplatins(e.g. ELOXATIN®); Paclitaxels (e.g. PAXENE®, TAXOL®); Pamidronates (e.g.AREDIA®); Pegademases (e.g. ADAGEN®); Pegaspargases (e.g. ONCASPAR®);Pegfilgrastims (e.g. NEULASTA®); Pentostatins (e.g. NIPENT®);Pipobromans (e.g. VERCYTE®); Plicamycin/Mithramycins (e.g. MITHRACIN®);Porfimer sodiums (e.g. PHOTOFRIN®); Procarbazines (e.g. MATULANE®);Quinacrines (e.g. ATABRINE®); Rasburicases (e.g. ELITEK®); Rituximabs(e.g. RITUXAN®); Sargramostims (e.g. PROKINE®); Streptozocins (e.g.ZANOSAR®); Sunitinib Malates (e.g. SUTENT®); Talcs (e.g. SCLEROSOL®);Tamoxifens (e.g. NOLVADEX®); Temozolomides (e.g. TEMODAR®);Teniposides/VM-26s (e.g. VUMON®); Testolactones (e.g. TESLAC®);Thioguanines including, 6-thioguanine (6-TG); Thiotepas (e.g.THIOPLEX®); Topotecans (e.g. HYCAMTIN®); Toremifenes (e.g. FARESTON®);Tositumomabs (e.g. BEXXAR®); Trastuzumabs (e.g. HERCEPTIN®);Tretinoins/ATRA (e.g. VESANOID®); Uracil Mustards; Valrubicins (e.g.VALSTAR®); Vinblastines (e.g. VELBAN®); Vincristines (e.g. ONCOVIN®);Vinorelbines (e.g. NAVELBINE®); and Zoledronates (e.g. ZOMETA®).

4. Packaging and Articles of Manufacture

Also provided are articles of manufacture containing packagingmaterials, any pharmaceutical composition or combination providedherein, and a label that indicates that the compositions andcombinations are to be used for treatment of side effects associatedwith administration of an anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzyme, and/or for treating a hyaluronan-associateddisease or condition. Exemplary of articles of manufacture arecontainers including single chamber and dual chamber containers. Thecontainers include, but are not limited to, tubes, bottles and syringes.The containers can further include a needle for subcutaneousadministration.

In one example, pharmaceutical composition contains the corticosteroidand no second agent or treatment. In another example, the article ofmanufacture contains pharmaceutical compositions containing thecorticosteroid and the anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzyme. In this example, the corticosteroid and theanti-hyaluronan agent, for example a PEGylated hyaluronan degradingenzyme, can be provided together or separately, for packaging asarticles of manufacture. In another example, the article of manufacturecontains the corticosteroid, anti-hyaluronan agent (e.g. a PEGylatedhyaluronan degrading enzyme) and a second agent or agents or treatmentor treatments. In this example, the corticosteroid, anti-hyaluronanagent (e.g. a PEGylated hyaluronan degrading enzyme) and a second agentor agents or treatment or treatments, can be provided together orseparately, for packaging as articles of manufacture.

In one example, the pharmaceutical composition contains theanti-hyaluronan agent (e.g. a PEGylated hyaluronan degrading enzyme),and no second agent or treatment. In another example, the article ofmanufacture contains the anti-hyaluronan agent (e.g. a PEGylatedhyaluronan degrading enzyme) and a second agent or agents or treatmentor treatments. In this example, the pharmaceutical compositions of asecond agent and an anti-hyaluronan agent, for example a PEGylatedhyaluronan degrading enzyme, such as a PEGylated hyaluronidase, can beprovided together or separately, for packaging as articles ofmanufacture.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment.

The choice of package depends on the corticosteroid, such as aglucocorticoid and other agents, and whether such compositions will bepackaged together or separately. In general, the packaging isnon-reactive with the compositions contained therein. In other examples,some of the components can be packaged as a mixture. In other examples,all components are packaged separately. Thus, for example, thecomponents can be packaged as separate compositions that, upon mixingjust prior to administration, can be directly administered together.Alternatively, the components can be packaged as separate compositionsfor administration separately.

The components can be packaged in a container. The components areseparately packaged in the same container. Generally, examples of suchcontainers include those that have an enclosed, defined space thatcontains glucocorticoid, and a separate enclosed, defined spacecontaining the other components or component such that the subsequentareas are separated by a readily removable membrane which, upon removal,permits the components to mix. Any container or other article ofmanufacture is contemplated, so long as the glucocorticoid is separatedfrom the other components prior to administration. For suitableembodiments see e.g., containers described in U.S. Pat. Nos. 3,539,794and 5,171,081.

Selected compositions including articles of manufacture thereof also canbe provided as kits. Kits can include a pharmaceutical compositiondescribed herein and an item for administration provided as an articleof manufacture. The kit can, optionally, include instructions forapplication including dosages, dosing regimens and instructions formodes of administration. Kits also can include a pharmaceuticalcomposition described herein and an item for diagnosis.

G. Methods of Assessing Activity and Effects of Anti-Hyaluronan Agentsand Corticosteroids

Provided herein are methods for treating a subject with acorticosteroid, for example a glucocorticoid such as dexamethasone, toameliorate, and/or eliminate adverse effects resulting from anadministered anti-hyaluronan agent. For example, systemic administrationof PEGylated hyaluronidase is associated with musculoskeletal sideeffects including, for example, muscle and joint pain, stiffness ofupper and lower extremities, cramping, myositis, muscle soreness andtenderness over the entire body, weakness, fatigue and/or a decrease inrange of motion at knee and elbow joints. In such methods, thecorticosteroid is generally administered to ameliorate side effectswithout eliminating the activities of the anti-hyaluronan agent toreduce hyaluronan by inhibiting its synthesis or degrading hyaluronan.It is within the level of one of skill in the art to assess whethercorticosteroids ameliorate such side effects, including withoutinterfering with the activity of an anti-hyaluronan agent, such as aPEGylated hyaluronidase. For example, various animal models and clinicalstudies in humans can be performed. In addition to assessing theamelioration of side effects, efficacy, tolerability and pharmacokineticstudies of an anti-hyaluronan agent can be performed in the presence orabsence of varying doses of corticosteroid.

In particular, PEGylated hyaluronan is a therapeutic agent either alone,or in combination with secondary agents such as chemotherapeutic drugs,for the treatment of hyaluronan-associated diseases and conditions, inparticular cancers (see for example, US 2010/0003238 and WO09/128,917).Hence, amelioration of side effects of PEGylated hyaluronidase withcorticosteroids permits the use of PEGylated hyaluronidase in suchtreatments while minimizing the systemic, for example musculoskeletal,side effects of the PEGylated hyaluronidase. Studies, including inanimal models of a hyaluronan-associated disease such as cancer, can beperformed to assess the efficacy of PEGylated hyaluronidase, alone or incombination with chemotherapeutic agents, and in the presence or absenceof corticosteroid treatment.

1. Methods to Assess Side Effects

In vivo assays can be used to assess the efficacy of corticosteroids onthe amelioration or elimination of the musculoskeletal side effects.Side effects that can be assessed include, for example, muscle and jointpain, stiffness of upper and lower extremities, cramping, myositis,muscle soreness and tenderness over the entire body, weakness, fatigueand/or a decrease in range of motion at knee and elbow joints. Assays toassess side effects can include animal models wherein the animal can beobserved for reduced movement, behavior or posture changes, radiographicfindings, histopathological changes and other notable clinicalobservations. Other assays can include clinical trials in human subjectswherein patients can be questioned regarding symptoms, assessed byphysical examination, imaging (for example by MRI or PET) or byradiologic evaluation. Amelioration of a side effect caused byadministration of an anti-hyaluronan agent is observed when the sideeffect is ameliorated, eliminated, lessened or reduced in the presenceof the corticosteroid compared to in its absence.

In such examples, the dose of anti-hyaluronan agent and/orcorticosteroid can be varied to identify the optimal or minimal doserequired to achieve activity while ameliorating side effects. Suchstudies are within the level of one of skill in the art. Further, thedosage regime can be varied. For example, studies can be performed usinga dosage schedule of anti-hyaluronan agent monthly, biweekly, once aweek, twice a week, three times a week, four times a week or more.Further, the corticosteroid can be administered prior to, concurrentlyand/or subsequent to administration of the anti-hyaluronan agent. TheExamples exemplify such studies in animal models and human patients.

For example, in vivo animal models can be utilized to assess the abilityof corticosteroids, such as dexamethasone, to ameliorate or eliminatethe side effects associated with anti-hyaluronan agent administration.Animal models can include non-human primates such as cynomolgus monkeysor rhesus macaques, dogs, for example beagle dogs, or any other animalthat exhibits adverse side effects in response to PEGylatedhyaluronidase treatment. The animal models can be dosed with ananti-hyaluronan agent in the presence or absence of corticosteroid andmusculoskeletal effects observed or measured.

For example, animals such as cynomolgus monkeys, beagles or othersimilar animal model capable of observable or measurable musculoskeletalevents can be treated with an anti-hyaluronan agent in the presence orabsence of corticosteroid. In one example, a group of animals, forexample cynomolgus monkeys or beagles, is administered with ananti-hyaluronan agent alone, for example a PEGylated hyaluronidase, suchas by intravenous administration. For example, administration can betwice weekly. Treatment can continue until changes in limb jointrange-of-motion are observed at the knee and elbow joints or stiffnessor reduced mobility is observed. Then, another group of animals can betreated with the anti-hyaluronan agent and corticosteroid administered,such as by oral doses of dexamethasone or other corticosteroid, given onthe same day as the anti-hyaluronan agent administration. The groups ofanimals can then be compared for example, via physical examination ofjoint range-of-motion or other reduced mobility, histopathology of thejoints, palpation for stiffness, or imaging known to those of skill inthe art, to assess the ability of the corticosteroid, such asdexamethasone, to ameliorate the anti-hyaluronan agent-mediatedmusculoskeletal side effects. Dose, dosing frequency, route ofadministration, and timing of dosing of corticosteroid, such asdexamethasone, can be varied to optimize the effectiveness of thecorticosteroid.

In another example, the efficacy of corticosteroids such asdexamethasone on the amelioration or elimination of the adverse sideeffects associated with anti-hyaluronan agent administration can beassessed in human patients with solid tumors. For example, a clinicaltrial can be designed to examine the ability of corticosteroid toameliorate and/or eliminate anti-hyaluronan agent-mediated adverseevents including, but not limited to any one or more of the following:muscle and joint pain/stiffness of upper and lower extremities,cramping, muscle, myositis muscle soreness and tenderness over theentire body, weakness and fatigue. Patients can be treated withanti-hyaluronan agent with or without co-treatment with a corticosteroidsuch as dexamethasone. During and after administration ofanti-hyaluronan agent, side effects of both treatment groups can beassessed. A physician can determine the severity of the symptoms byphysical examination of the subject including for example, patientcomplaints, vital signs, changes in body weight, 12-lead ECG,echocardiogram, clinical chemistry, or imaging (MRI, PET or radiologicevaluation). The severity of symptoms can then be quantified using theNCI Common Terminology Criteria for Adverse Events (CTCAE) gradingsystem. The CTCAE is a descriptive terminology utilized for AdverseEvent (AE) reporting. A grading (severity) scale is provided for each AEterm. The CTCAE displays Grades 1 through 5, with clinical descriptionsfor severity for each adverse event based on the following generalguideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3 (SevereAE); Grade 4 (Life-threatening or disabling AE); and Grade 5 (Deathrelated to AE). The ability of a corticosteroid to ameliorate adverseside effects associated with administration of an anti-hyaluronan agentcan be measured by the observation of a reduction in grading or severityon the CTCAE scale in one or more adverse side effects in subjectstreated with the anti-hyaluronan agent and corticosteroid as compared tosubjects treated with the anti-hyaluronan alone, i.e., the severity ofthe side effects, is reduced from Grade 3 to Grade 1 or Grade 2. This isexemplified in the Examples herein.

In another example, clinical trials can be designed to assesstolerability by escalating the dose of anti-hyaluronan agent andassessing the dose-limiting toxicity. In such an example, a maximumtolerated dose of anti-hyaluronan agent that can be tolerated in thepresence of an ameliorating agent such as a corticosteroid can bedetermined. Treatment regimens can include a dose escalation whereineach enrolled patient receives a higher dose of anti-hyaluronan agent atthe same dose level of corticosteroid. Patients can be monitored foradverse events to determine the highest dose of anti-hyaluronan agentthat can be administered with a corticosteroid before side effects areno longer tolerated. Tolerability can be measured based on the severityof symptoms emerging during and after treatment. Doses ofanti-hyaluronan agent can be escalated until adverse effects reach apredetermined level, for example, Grade 3. Dosing regimens can alsoinclude a tapering of the amount of corticosteroid administered toexamine the continued need for corticosteroid and the possibility ofacclimation to the anti-hyaluronan agent with respect to resulting sideeffects.

2. Anti-Hyaluronan Activity

In addition to assays to assess the effect of corticosteroids onameliorating the side effects of an anti-hyaluronan agent, other assayscan be performed separately or in conjugation with those mentioned aboveto assess the effects of corticosteroids on hyaluronan inhibition ordegradation activity. Such assays can include, but are not limited to,measuring amounts of hyaluronan in tissue or tumor biopsies or solublehyaluronan in plasma, measurements of hyaluronan catabolites in blood orurine, measurements of hyaluronidase activity in plasma, or measurementsof interstitial fluid pressure, vascular volume or water content intumors. Other assays such as measurements of pharmacokinetics, methodsfor which are well known to those of skill in the art, can be used toassess the effects of corticosteroids on anti-hyaluronan agentpharmacokinetic parameters.

a. Assays to Assess the Activity of a Hyaluronan Degrading Enzyme

The activity of a hyaluronan degrading enzyme can be assessed usingmethods well known in the art. For example, the USP XXII assay forhyaluronidase determines activity indirectly by measuring the amount ofundegraded hyaluronic acid, or hyaluronan, (HA) substrate remainingafter the enzyme is allowed to react with the HA for 30 min at 37° C.(USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention,Inc, Rockville, Md.). A Hyaluronidase Reference Standard (USP) orNational Formulary (NF) Standard Hyaluronidase solution can be used inan assay to ascertain the activity, in units, of any hyaluronidase. Inone example, activity is measured using a microturbididy assay. This isbased on the formation of an insoluble precipitate when hyaluronic acidbinds with albumin. The activity is measured by incubating hyaluronidaseor a sample containing hyaluronidase, for example blood or plasma, withsodium hyaluronate (hyaluronic acid) for a set period of time (e.g. 10minutes) and then precipitating the undigested sodium hyaluronate withthe addition of acidified serum albumin. The turbidity of the resultingsample is measured at 640 nm after an additional development period. Thedecrease in turbidity resulting from hyaluronidase activity on thesodium hyaluronate substrate is a measure of hyaluronidase enzymaticactivity.

In another example, hyaluronidase activity is measured using amicrotiter assay in which residual biotinylated hyaluronic acid ismeasured following incubation with hyaluronidase or a sample containinghyaluronidase, for example, blood or plasma (see e.g. Frost and Stern(1997) Anal. Biochem. 251:263-269, U.S. Patent Publication No.20050260186). The free carboxyl groups on the glucuronic acid residuesof hyaluronic acid are biotinylated, and the biotinylated hyaluronicacid substrate is covalently coupled to a microtiter plate. Followingincubation with hyaluronidase, the residual biotinylated hyaluronic acidsubstrate is detected using an avidin-peroxidase reaction, and comparedto that obtained following reaction with hyaluronidase standards ofknown activity. Other assays to measure hyaluronidase activity also areknown in the art and can be used in the methods herein (see e.g. Delpechet al., (1995) Anal. Biochem. 229:35-41; Takahashi et al., (2003) Anal.Biochem. 322:257-263).

The ability of an active hyaluronan degrading enzyme, such as a modifiedsoluble hyaluronidase (eg PEGylated rHuPH20) to act as a spreading ordiffusing agent also can be assessed. For example, trypan blue dye canbe injected, such as subcutaneously or intradermally, with or without ahyaluronan degrading enzyme into the lateral skin on each side of nudemice. The dye area is then measured, such as with a microcaliper, todetermine the ability of the hyaluronan degrading enzyme to act as aspreading agent (see e.g. U.S. Published Patent No. 20060104968).

The above assays can be performed using a hyaluronan degrading enzyme inthe presence or absence of a corticosteroid or using the blood or plasmaof a patient or animal treated with hyaluronidase with or without acorticosteroid.

b. Assays in Animal Models

Animal models of hyaluronan-associated diseases, disorders or conditionscan be utilized to assess the in vivo affect of administration of ananti-hyaluronan agent, such as a modified hyaluronidase or PEGylatedhyaluronidase, with or without co-administration of a corticosteroid.Another agent, such as a chemotherapeutic agent can also be included inthe assessment of activity. Exemplary hyaluronan-associated diseases forwhich an appropriate animal model can be utilized include solid tumors,for example, late-stage cancers, metastatic cancers, undifferentiatedcancers, ovarian cancer, in situ carcinoma (ISC), squamous cellcarcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lungcancer, breast cancer, colon cancer and other cancers. Also exemplary ofhyaluronan-associated diseases and disorders are inflammatory diseases,disc pressure, cancer and edema, for example, edema caused by organtransplant, stroke, brain trauma or other injury.

Animal models can include, but are not limited to, mice, rats, rabbits,dogs, guinea pigs and non-human primate models, such as cynomolgusmonkeys or rhesus macaques. In some examples, immunodeficient mice, suchas nude mice or SCID mice, are transplanted with a tumor cell line froma hyaluronan-associated cancer to establish an animal model of thatcancer. Exemplary cell lines from hyaluronan-associated cancers include,but are not limited to, PC3 prostate carcinoma cells, BxPC-3 pancreaticadenocarcinoma cells, MDA-MB-231 breast carcinoma cells, MCF-7 breasttumor cells, BT474 breast tumor cells, Tramp C2 prostate tumor cells andMat-LyLu prostate cancer cells, and other cell lines described hereinthat are hyaluronan associated, e.g. contain elevated levels ofhyaluronan. Anti-hyaluronan agents can then be administered to theanimal with or without a corticosteroid such as dexamethasone, to assessthe effects of the corticosteroid on anti-hyaluronan activity bymeasuring, for example, hyaluronan levels or content. Hyaluronan contentcan be measured by staining tumor tissue samples for hyaluronan or bymeasuring soluble hyaluronan levels in plasma. Other measurements ofanti-hyaluronan activity include the assessment of tumor volume,formation or size of halos, interstitial fluid pressure, water contentand/or vascular volume. Assays such as those mentioned above aredemonstrated in Example 9.

In other examples, dogs such as beagle dogs, can be treated with ananti-hyaluronan agent in the presence or absence of a corticosteroid,such as dexamethasone. Tissues such as skin or skeletal muscle tissueare biopsied and stained for hyaluronan and evaluated visually. Tissuesfrom animals treated with an anti-hyaluronan agent alone are thencompared to tissues from aminals treated with the anti-hyaluronan agentand corticosteroid to measure the effect of the corticosteroid onanti-hyaluronan activity. These assays are demonstrated in Example 8.

c. Assays in Humans

Clinical trials such as those described in Section G1 above, performedto assess the ability of corticosteroids to ameliorateanti-hyaluronan-mediated adverse effects, can concurrently be used toassay the activity of anti-hyaluronan agent in the presence of acorticosteroid. Assays to measure anti-hyaluronan activity in patientswith solid tumors, treated for example, with an anti-hyaluronan agentwith or without corticosteroids, with escalating doses ofanti-hyaluronan agent and a fixed amount of corticosteroid, or with afixed amount of anti-hyaluronan agent and tapering doses ofcorticosteroid, can be performed. These assays can include tumor tissuebiopsy assays where tumor biopsies are taken before treatment, duringtreatment and after treatment. Tissues are stained to measure hyaluronanlevels in the tumor to assess the activity of the administeredanti-hyaluronan agent. Stained tumor tissues biopsied before, during andafter treatment can be compared to evaluate anti-hyaluronan activity inthe presence of corticosteroids.

In another example, blood and urine can be collected at different timepoints throughout patient treatment and assayed for catabolites ofhyaluronan. The presence of catabolites is indicative of the degradationof hyaluronan and is thus a measure of the activity of hyaluronidase.Plasma enzyme also can be assessed and measured over time followingadministration. The Examples exemplify these assays.

Additional methods of assessing the anti-hyaluronan activity includeassays that assess the diffusion of water in tissues. As discussedelsewhere herein, tissues that accumulate hyaluronan generally have ahigher interstitial fluid pressure than normal tissue due to theconcomitant accumulation of water. Thus, tissues that accumulate HA,such as tumors, have high interstitial fluid pressure, which can bemeasured by various methods known in the art. For example, diffusionMRI, such as ADC MRI or DCE MRI, can be used. Diffusion of water can beassessed by these procedures, and is directly correlated to presence ofhyaluronan-rich tissues, such as solid tumors (see e.g. Chenevert et al.(1997) Clinical Cancer Research, 3:1457-1466). For example, tumors thataccumulate hyaluronan have a distinguishable increase in ADC MRI or DCEMRI because of increased perfusion. Such assays can be performed in thepresence and absence of an anti-hyaluronan agent with or withoutcorticosteroid, and results compared. Methods of measuring diffusion area useful measure of assessing cellular changes following such therapies.

d. Pharmacokinetics

Pharmacokinetic studies can be performed using animal models or can beperformed during clinical studies with patients to assess the effect ofco-administration of a corticosteroid on the pharmacokinetic propertiesof a modified hyaluronan degrading enzyme, such as a modifiedhyaluronidase. Animal models include, but are not limited to, mice,rats, rabbits, dogs, guinea pigs and non-human primate models, such ascynomolgus monkeys or rhesus macaques. In some instances,pharmacokinetic studies are performed using healthy animals. In otherexamples, the studies are performed using animal models of a disease forwhich therapy with hyaluronan is considered, such as animal models ofany hyaluronan-associated disease or disorder.

The pharmacokinetic properties of an anti-hyaluronan agent, such as amodified hyaluronidase, in the presence of a corticosteroid, can beassessed by measuring such parameters as the maximum (peak)concentration (C_(max)), the peak time (i.e. when maximum concentrationoccurs; T_(max)), the minimum concentration (i.e. the minimumconcentration between doses; C_(min)), the elimination half-life(T_(1/2)) and area under the curve (i.e. the area under the curvegenerated by plotting time versus concentration; AUC), followingadministration. In instances where the modified hyaluronidase might beadministered subcutaneously, the absolute bioavailability of thehyaluronidase is determined by comparing the area under the curve ofhyaluronidase following subcutaneous delivery (AUC_(sc)) with the AUC ofhyaluronidase following intravenous delivery (AUC_(iv)). Absolutebioavailability (F), can be calculated using the formula:F=([AUC]_(sc)×dose_(sc))/([AUC]_(iv)×dose_(iv)).

A range of doses and different dosing frequency of dosing can beadministered in the pharmacokinetic studies to assess the effect ofincreasing or decreasing concentrations of the corticosteroid, such asdexamethasone and/or anti-hyaluronan agent (e.g. PEGylated rHuPH20) inthe dose. Pharmacokinetic properties of anti-hyaluronan agents, such asbioavailability, also can be assessed with or without co-administrationof corticosteroid. For example, dogs, such as beagles, can beadministered an anti-hyaluronan agent, such as a PEGylatedhyaluronidase, alone, or together with a corticosteroid, using one ormore routes of administration. Such studies can be performed to assessthe effect of co-administration of corticosteriods with anti-hyaluronanagent on pharmacokinetic properties. Additionally, the effect ofco-administration of an anti-hyaluronan agent, such as a hyaluronidase,with another agent, such as a chemotherapeutic, in the presence orabsence of a corticosteroid, on the pharmacokinetic and pharmacodynamicproperties of that agent also can be assessed in vivo using animal modeland/or human subjects, such as in the setting of a clinical trial, asdiscussed above.

H. Use of Anti-Hyaluronan Agents in Treating Hyaluronan-AssociatedConditions, Diseases and Disorders

Anti-Hyaluronan agents, such as a PEGylated hyaluronan degrading enzyme,can be used to treat hyaluronan-associated diseases or conditions, aloneor in combination with therapeutic agents. In particular, modifiedhyaluronan degrading enzymes, such as a PEGPH20, has anti-tumor activityalone or in combination with chemotherpeutic agents (see e.g. U.S.published application No. US20100003238 and International publishedapplication No. WO2009128917).

As found herein, anti-tumor activity is observed at doses far lower thanpreviously contemplated that do not achieve or maintain a detectablelevel of hyaluronidase enzyme in the plasma. Hence, the extent ofadverse side effects, such as musculoskeletal side effects, is not assevere as higher doses of enzyme. At these lower doses, the enzymeexhibits pharmacokinetic and pharmacodynamic properties that correlateto a reduction in tumor associated hyaluronan (HA), elevation in plasmahyaluronan, and favorable changes in tumor volumes and incidence.

Hence, provided herein is a method or use for treating ahyaluronan-associated disease or condition, such as a cancer, byadministering a polymer-modified hyaluronan-degrading enzyme, such as apolymer-modified hyaluoronidase for example a PH20 (e.g. PEGPH20) to apatient in an amount less than 20 μg/kg, for example 0.01 μg/kg to 15μg/kg, 0.05 μg/kg to 10 μg/kg, 0.75 μg/kg to 7.5 μg/kg or 1.0 μg/kg to3.0 μg/kg, such as at or about 0.01 μg/kg (of the subject), 0.02 μg/kg,0.03 μg/kg, 0.04 μg/kg, 0.05 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 2.5μg/kg, 3.0 μg/kg, 3.5 μg/kg, 4.0 μg/kg, 4.5 μg/kg, 5.0 μg/kg, 5.5 μg/kg,6.0 μg/kg, 7.0 μg/kg, 7.5 μg/kg, 8.0 μg/kg, 9.0 μg/kg, 10.0 μg/kg, 12.5μg/kg or 15 μg/kg. For example, a polymer-modified hyaluronan-degradingenzyme, such as a PEGylated hyaluronidase (e.g. PH20), provided herein,for example, PEGPH20, can be administered at or about 1 Unit/kg to 1000Units/kg, 1 Units/kg to 500 Units/kg or 10 Units/kg to 50 Units/kg.Generally, where the specific activity of the modified hyaluronidase isor is about 20,000 U/mg to 60,000 U/mg, generally at or about 35,000U/mg, 200 Units to 50,000 (U) is administered, such as 200 U, 300 U; 400U; 500 U; 600 U; 700 U; 800 U; 900 U; 1,000 U; 1250 U; 1500 U; 2000 U;3000 U; 4000 U; 5,000 U; 6,000 U; 7,000 U; 8,000 U; 9,000 U; 10,000 U;20,000 U; 30,000 U; 40,000 U; or 50,000 U is administered. Typically,volumes for injection or infusion are less than 50 mL, such as from ator about 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL,10 mL, 15 mL, 20 mL, 30 mL, 40 mL or 50 mL.

For the treatment of a hyaluronan-associated disease or condition, suchas a cancer, the polymer-modified hyaluronan-degrading enzyme can beadministered systemically, for example, intravenously (IV),intramuscularly, or by any another systemic route. In particularexamples, the lower doses can be given locally. For example, localadministration of a hyaluronan-degrading enzyme, such as a PEGylatedhyaluronan degrading enzyme for example a PEGylated hyaluronidase (e.g.PH20) includes intratumoral administration, arterial injection (e.g.hepatic artery), intraperitoneal administration, intravesicaladministration and other local routes used for cancer therapy that canincrease local action at a lower absolute dose. In the methods hereinfor treating a hyaluronan-associated disease or condition, such as acancer, the enzyme can be administered alone or in combination withtherapeutic agents. Further, as provided in other methods herein,corticosteroids can be administered to ameliorate any side effects oradverse events associated with the hyaluronan-degrading enzyme.

In the methods herein of treating a hyaluronan-associated disease orcondition, the amounts of a polymer-modified hyaluronan-degrading enzymeas set forth above are administered periodically over a cycle ofadministration. For example, periodic administration of apolymer-modified hyaluronan-degrading enzyme can be twice weekly, onceweekly or once every 21 days. The length of time of the cycle ofadministration can be empirically determined, and is dependent on thedisease to be treated, the severity of the disease, the particularpatient, and other considerations within the level of skill of thetreating physician. The length of time of treatment with a modifiedhyaluronidase enzyme can be one week, two weeks, one months, severalmonths, one year, several years or more. For example, a modifiedhyaluronidase enzyme can be administered twice weekly over a period of ayear or more. In some examples, the periodic frequency of administrationin subsequent cycles of administration can be reduced. For example, amodified hyaluronidase enzyme can be administered twice weekly for 4weeks, and then administered once weekly over a period of a year ofmore. If disease symptoms persist in the absence of discontinuedtreatment, treatment can be continued for an additional length of timeand/or the period of frequency of administration can be increased. Overthe course of treatment, evidence of disease and/or treatment-relatedtoxicity or side effects can be monitored. If side effects are observed,a corticosteroid agent can be included in the dosage regime as describedherein.

In addition, the cycle of administration can be tailored to add periodsof discontinued treatment in order to provide a rest period fromexposure to the enzyme. The length of time for the discontinuation oftreatment can be for a predetermined time or can be empiricallydetermined depending on how the patient is responding or depending onobserved side effects. For example, the treatment can be discontinuedfor one week, two weeks, one month or several months. Generally, theperiod of discontinued treatment is built into a cycle of dosing regimefor a patient. For example, an exemplary dosing regime is a treatmentcycle of 28 days, with the modified enzyme administered for the first 3weeks, twice weekly, followed by a one week without dosing. Thus, forexample, a patient can be dosed with modified enzyme on days 1, 4, 8,11, 15 and 18, followed by a one-week of discontinued treatment, overthe course of the 28-day cycle. As noted above, the cycle ofadministration can be for any desired length of time. Hence, the 28-daycycle of administration can be repeated for any length of time. It iswithin the level of skill of the treating physician to adopt a cycle ofadministration and dosing regime that meets the needs of the patientdepending on personal considerations specific to the patient and diseaseto be treated.

In particular examples, a single dosage administration of apolymer-modified hyaluronan-degrading enzyme, such as a PEGylatedhyaluronidase (e.g. PH20), provided herein, for example, PEGPH20, is anamount in a range between or about between 0.5 μg to 1450 μg or 150Units (U) to 45,000 Units. For example, a single dosage administrationis of an amount in a range between or about between 0.75 μg to 1125 μg;3.75 μg to 750 μg; 56 μg to 565 μg; or 75 μg to 225 μg. In otherexamples, a single dosage administration is of an amount in a rangebetween or about between 24 Units (U) to 36,000 U; 120 U to 24,000 U;1500 U to 18,000 U; or 2400 U to 7200 U. For example, provided hereinare uses of a hyaluronan-degrading enzyme that is administered to asubject as a unit dosage of an amount in a range between or aboutbetween 0.5 μg to 1450 μg or 150 Units (U) to 45,000 Units at afrequency of at least once a week for a cycle of at least 4 weeks. Thefrequency can be at least twice a week or once a week. It is understoodthat the cycle of administration for treating a hyaluronan-associateddisease or condition can be repeated a plurality of times depending onthe particular needs of the patient.

In some examples provided herein, using the methods herein,corticosteroids can be administered in combination with ananti-hyaluronan agent for the treatment or use in treatment of ahyaluronan-associated disease or condition, such as in the treatment ofpatients with advance solid tumors. In some examples, chemotherapeuticagents or other anti-cancer agents also can be used in the therapy. Thetherapeutic uses described below are exemplary and do not limit theapplications of the methods described herein. It is understood that inthe description below, a corticosteroid, such as a glucocorticoid, forexample dexamethasone, can be used in combination with ananti-hyaluronan agent in the methods in order to reduce or amelioratemusculoskeletal side effects or other side effects. Dosages and route ofadministration are described above in Section F.

The provided methods include methods for use of the hyaluronan-degradingenzymes to treat any hyaluronan-associated disease or condition,including, but not limited to, one that is associated with highinterstitial fluid pressure, a cancer and in particular a hyaluronanrich cancer, edema, disc pressure, an inflammatory disease, and otherdiseases associated with hyaluronan. A hyaluronan-associated conditionsand diseases are diseases and conditions in which hyaluronan levels areelevated as cause, consequence or otherwise observed in the disease orcondition. In some cases, hyaluronan-associated diseases and conditionsare associated with increased interstitial fluid pressure, decreasedvascular volume, and/or increased water content in a tissue, such as atumor. Exemplary hyaluronan-associated diseases and conditions that canbe treated using the provided enzymes, compositions and methods,include, but are not limited to, hyaluronan-rich cancers, for example,tumors, including solid tumors such as late-stage cancers, a metastaticcancers, undifferentiated cancers, ovarian cancer, in situ carcinoma(ISC), squamous cell carcinoma (SCC), prostate cancer, pancreaticcancer, non-small cell lung cancer, breast cancer, colon cancer andother cancers.

Elevated levels of hyaluronan are assocated with numerous inflammatorydiseases, including virtually all disease processes involvinginflammation. Such diseases and conditions include, but are not limitedto, rheumatoid arthritis, periodontitis, scleroderma, psoriasis,atherosclerosis, chronic wounds, Crohn's disease, ulcerative colitis andinflammatory bowel disease. A mechanism for these elevated levels is dueto regulation of HA synthase genes by inflammatory mediators, such asIL-1β (Ducale et al. (2005) Am. J. Physiol. Gatrointest. Liver Physiol.,289:G462-G470). In addition, hyaluronan itself is able to interact withand activate various leukocytes, thereby exacerbating the inflammation(see e.g. Ducale et al. 2005; Jiang et al. (2007) Annu. Rev. Cell. Dev.Biol., 23:435-61). Hence, other hyaluronan-associated diseases andconditions that can be treated with anti-hyaluronan agents includeinflammatory diseases and conditions, including but not limited to,Rheumatoid arthritis, scleroderma, periodontitis, psoriasis,atherosclerosis, chronic wounds, Crohn's disease, ulcerative colitis andinflammatory bowel disease.

Also exemplary of hyaluronan-associated diseases and conditions arediseases that are associated with elevated interstitial fluid pressure,such as diseases associated with disc pressure, and edema, for example,edema caused by organ transplant, stroke, brain trauma or other injury.Exemplary hyaluronan-associated diseases and conditions include diseasesand conditions associated with elevated interstitial fluid pressure,decreased vascular volume, and/or increased water content in a tissue,including cancers, disc pressure and edema. In one example, treatment ofthe hyaluronan-associated condition, disease or disorder includesamelioration, reduction, or other beneficial effect on one or more ofincreased interstitial fluid pressure (IFP), decreased vascular volume,and increased water content in a tissue.

Typically, hyaluronan-associated diseases and conditions are associatedwith elevated hyaluronan expression in a tissue, cell, or body fluid(e.g. tumor tissue or tumor-associated tissue, blood, or interstitialspace) compared to a control, e.g. another tissue, cell or body fluid.The elevated hyaluron expression can be elevated compared to a normaltissue, cell or body fluid, for example, a tissue, cell or body fluidthat is analogous to the sample being tested, but isolated from adifferent subject, such as a subject that is normal (i.e. does not havea disease or condition, or does not have the type of disease orcondition that the subject being tested has), for example, a subjectthat does not have a hyaluronan-associated disease or condition. Theelevated hyaluronan expression can be elevated compared to an analogoustissue from another subject that has a similar disease or condition, butwhose disease is not as severe and/or is not hyaluronan-associated orexpresses relatively less hyaluronan and thus is hyaluronan-associatedto a lesser degree. For example, the subject being tested can be asubject with a hyaluronan-associated cancer, where the HA amounts in thetissue, cell or fluid are relatively elevated compared to a subjecthaving a less severe cancer, such as an early stage, differentiated orother type of cancer. In another example, the cell, tissue or fluidcontains elevated levels of hyaluronan compared to a control sample,such as a fluid, tissue, extract (e.g. cellular or nuclear extract),nucleic acid or peptide preparation, cell line, biopsy, standard orother sample, with a known amount or relative amount of HA, such as asample, for example a tumor cell line, known to express relatively lowlevels of HA, such as exemplary tumor cell lines described herein thatexpress low levels of HA, for example, the HCT 116 cell line, the HT29cell line, the NCI H460 cell line, the DU145 cell line, the Capan-1 cellline, and tumors from tumor models generated using such cell lines (see,e.g. Example 17A).

Typically, the hyaluronan-associated disease or condition is associatedwith increased HA expression, for example, in a diseased tissue, forexample, a tumor. In one example, HALOs (pericellular matrix regionsthat are rich in proteoglycans, including hyaluronan) form in a tissueof the subject, for example, in a diseased tissue. In another example,the presence of HALOs is detected in an in vitro culture of cells from atissue of the subject, for example, a diseased tissue.

In one example, the hyaluronan-associated condition, disease or disorderis associated with increased interstitial fluid pressure, decreasedvascular volume, or increased water content in a tissue. In one example,treatment of the hyaluronan-associated condition, disease or disorderincludes amelioration, reduction, or other beneficial effect on one ormore of increased interstitial fluid pressure (IFP), decreased vascularvolume, and increased water content in a tissue. The therapeutic usesinclude treatment of a hyaluronan-associated disease or condition,including cancer treatment, reduction in tumor volume, increasedsensitivity to chemotherapy or other cancer treatment, enhancingbioavailability or delivery of a cancer treating or other treatingagent, decreasing interstitial fluid pressure, increasing vascularvolume, decreasing water content in a tissue in the subject, and othertreatments.

1. Selection of Subjects for Treatment and Assessing Treatment Effects

The methods include steps for selecting subjects for treatment withanti-hyaluronan agents and for assessing treatment effects, such asefficacy of treatment. Such methods include methods for detectinghyaluronan-associated disease markers, which include any indication thata subject has a hyaluronan-associated disease, that the subject islikely to respond to treatment by the anti-hyaluronan agent, and/or thata sample from the subject, such as a tissue, cell or fluid, containselevated hyaluronan expression. Exemplary assays for detecting markersare described below, and include assays for measuring HA expressionand/or relative HA expression in a sample from a subject, assays foranalyzing effects of anti-hyaluronan agents on a sample from thesubject, and assays for measuring readouts typically associated withcertain hyaluronan-associated diseases/conditions, such as lowhyaluronidase expression or activity, high interstitial fluid pressure,vascular volume and water content. In general, any known assay fordetection of proteins or nucleic acids in samples from subjects, or forassessing the effects of treatment on cells/tissues in vitro can beused.

Subjects selected for treatment in the methods provided herein includesubjects having elevated, aberrant or accumulated expression ofhyaluronan compared to subjects not having the disease or condition orcompared to normal tissues or samples that do not have elevated,aberrant or accumulated expression of HA. Any sample or tissue from asubject can be tested and compared to a normal sample or tissue.Hyaluronan levels can be measured from any source such as from a tissue(e.g. by biopsy), tumor, cells, or from blood, serum, urine or otherbody fluids. For example, as described elsewhere herein, profiles of HAdeposition in solid tumors have generally been categorized aspericellular or stromal. Elevated plasma levels of HA have been observedmost notably in patients with Wilm's tumor, mesothelioma and livermetastases. Thus, depending on the disease or condition, a differentsample can be measured for hyaluronan levels. The choice of sample iswithin the level of one of skill in the art.

The assay used to measure hyaluronan levels is a function of the diseaseor condition and can be chosen based on the particular disease orcondition. One of skill in the art is familiar with methods of detectinghyaluronan, which include, but are not limited to, immunohistochemistrymethods, ELISA methods, as described in section (i) below.

In one example, the step for detecting markers is performed prior totreating a subject, for example, to determine whether the subject has ahyaluronan-associated condition or disease that will be amenable totreatment with an anti-hyaluronan agent. In this example, if the markeris detected (e.g. if it is determined that a cell, tissue or fluid fromthe patient contains elevated hyaluronan expression or is responsive tohyaluronan degrading enzyme), a treatment step is performed, where ahyaluronan-degrading enzyme is administered to the subject. In oneexample, when the marker is not detected (e.g. if it is determined thata cell, tissue or fluid from the patient contains normal or non-elevatedhyaluronan expression or is not responsive to an anti-hyaluronan agent)another treatment option may be selected.

In another example, the step for detecting markers is performed aftertreating a subject, or during the course of treatment of the subject,(e.g. treatment with the anti-hyaluronan agent (e.g. soluble modifiedhyaluronidase) (with or without a co-administered agent), for example,to determine whether the treatment with the anti-hyaluronan agent ishaving an effect on treating the disease or condition. In one suchexample, the marker is not detected or is detected at an amount orrelative level that is decreased compared to the amount/level prior totreatment, or compared to another sample, treatment is continued,another round of treatment is performed, or another treatment, such as acombination therapy, is initiated. In another such example, if themarker is detected at the same level as prior to treatment or anothersample, another treatment option may be selected.

a. Assays for Detection of Hyaluronan-Associated Disease Markers

The assays to detect markers of hyaluronan-associated diseases andconditions include assays to measure amount (e.g. relative amount) ofhyaluronan, HA synthase expression and/or hyaluronidase expression in atissue, cell and/or body fluid of a subject, for example, a tumor.Included amongst such assays are those that can detect HA expression,Hyaluronan synthase 2 (HAS2) expression, the presence of HALOs(pericellular matrix regions that are rich in proteoglycans, includinghyaluronan), and the presence of hyaluronan-degrading enzymes, such ashyaluronidases, for example, in samples from the subject.

Assays to detect protein and nucleic acid levels are well known in theart and can be used in the methods herein to measure hyaluronan,hyaluronan synthase or other protein and/or nucleic acid expression.Such assays include, but are not limited to, ELISA, SDS-PAGE, WesternBlot, PCR, RT-PCR, immunohistochemistry, histology and flow cytometry.For example, a sample from a subject, such as a tissue sample (e.g. abiopsy of a tumor from a patient or animal model, a stromal sample), afluid (e.g. blood, urine, plasma, saliva or other sample), a cell orcellular sample, or extract, or other sample, can be stained withanti-HA antibodies, for example, using histological staining, such asimmunohistochemistry (IHC) of fixed or frozen tissue sections, todetermine the presence and extent of hyaluronan in the tissue or sample,or immunofluorescent cellular staining, pull-down assays, and flowcytometry. In another example, the sample, e.g. biopsy, can be assayedby RT-PCR to assess the amount of HA mRNA.

Known methods for detection of hyaluronan-expression in cancer include,but are not limited to, the ELISA-like assay described in Lokeshwar etal., Cancer Res. 57: 773-777 (1997), for measuring HA levels in urine orbladder tissue extracts of subjects having bladder cancer. For theassay, urine or extracts are coated on microwell plates (umbilical cordHA used as a standard also is coated), followed by incubation (e.g. 16hours, room temperature) with a labeled (e.g. biotinylated) HA bindingprotein, such as those described herein, washed and the HA-bindingprotein bound to the wells quantified using an avidin-biotin detectionagent substrate. Such methods are well known in the art. In one example,the urine from a subject with an HA-associated bladder cancer containedHA levels that were elevated 2-9 fold compared to urine/extracts fromnormal patients (healthy subjects or subjects with other gastrourinarydiseases or conditions); thus the marker would be detected if the HAlevels in the urine was elevated compared to normal subjects, e.g.elevated from between at or about 2-fold and at or about 9-fold, e.g. ator about 2, 3, 4, 5, 6, 7, 8 or 9-fold elevation compared to normalsubject.

In a further example, hyaluronan expression and production in tumorcells in vitro can be assessed using any one of the methods describedabove. Similarly, Hyaluronan synthase 2 (HAS2) production and/orexpression by cells in vitro, ex vivo or in vivo also can be assayed by,for example, ELISA, SDS-PAGE, Western Blot, PCR, RT-PCR,immunohistochemistry, histology or flow cytometry.

In another example, the amount of hyaluronidase activity in a samplefrom the subject is determined, such as in the blood or plasma, forexample, such as with a turbidity assay.

In another example, a cell or other tissue from a patient is isolated,e.g. a tumor cell, and used in a study to determine whether the cell ortissue is responsive to treatment with the hyaluronan degrading enzymein vitro, for example, using a clonogenic assay or any other assay formeasuring growth, proliferation and/or survival of cells or tissues,such as tumor cells, in response to treatment. In one example, cancercells from a subject are seeded on a surface, such as an extracellularmatrix or protein mixture, such as the mixture sold under the trade nameMatrigel® (BD Biosciences). In this example, the hyaluronan-associatedmarker is the sensitivity of the cell or tissue to administration ofhyaluronan degrading enzyme. In this example, if any property, such asproliferation, growth or survival of the cells, is inhibited or blockedby addition of hyaluronan degrading enzyme, it is determined that thesubject may be amenable to treatment with hyaluronan degrading enzymecontaining compositions.

In addition to assays for determining hyaluronan expression levels,other assays can be used to select a subject for treatment, and/or toassess treatment efficacy and/or duration. Interstitial fluid pressure(IFP) can be measured using an appropriate probe or instrument. Forexample, a transducer-tipped catheter can be used to measure the IFP incancer tissues or other tissues of interest. The catheter is passedthrough the inner bore of a surgical needle, which is then inserted intothe center of the tumor. The needle is withdrawn while the catheter isheld in position. The IEP (mmHg) can then be measured using anappropriate data acquisition unit (see e.g. Ozerdem et al. (2005)Microvasc. Res. 70:116-120). Other methods to measure IFP include thewick-in-needle method (Fadnes et al. (1977) Microvasc. Res. 14:27-36).

Vascular volume can be measured by, for example, by ultrasound imaging.This method employs hyper-echoic microbubbles to provide the strongultrasound wave reflections that are detected. The microbubbles, wheninjected, such as intravenously, into a subject or animal model, becometrapped in the vascular space due to their size. Assays to assess tissuewater content, such as tumor tissue water content, also are known in theart. For example, samples from a tumor can be harvested, blotted,weighed and snap frozen before being lyophilized. The water weight isthen reported as the tissue wet weight to dry (i.e. lyophilized) weightratio.

The ability of a tumor cell to form pericellular matrices (halos) invitro can be assessed using a particle exclusion assay. Small particles(formalin-fixed red blood cells) can be added to low-density cultures oftumor cells in the presence of, for example, aggrecan, which is a largeaggregating chondroitin sulfate proteoglycan. After the particlessettle, the cultures can be viewed at 400× magnification to determinewhether any halos were formed by the tumor cells. This can arevisualized as areas around the cells from which the particles areexcluded.

b. Detection of Hyaluronan-Associated Markers Relative to ControlSamples

For any of the detection methods, the marker (e.g. HA expression,responsiveness to hyaluronan degrading enzyme, HA-synthase expression orhyaluronidase activity) typically is compared to a control sample, suchthat detection of the marker typically includes determining that thereadout is elevated or reduced compared to the control sample.

For example, the control sample can be another tissue, cell or bodyfluid, such as a normal tissue, cell or body fluid. For example, atissue, cell or body fluid that is analogous to the sample being tested,but isolated from a different subject, such as a subject that is normal(i.e. does not have a disease or condition, or does not have the type ofdisease or condition that the subject being tested has) can be tested.In another example, an analogous tissue from another subject that has asimilar disease or condition, but whose disease is not as severe and/oris not hyaluronan-associated or expresses relatively less hyaluronan canbe tested. For example, when the cell, tissue or fluid being tested is asubject having a cancer, it can be compared to a tissue, cell or fluidfrom a subject having a less severe cancer, such as an early stage,differentiated or other type of cancer. In another example, the controlsample is a fluid, tissue, extract (e.g. cellular or nuclear extract),nucleic acid or peptide preparation, cell line, biopsy, standard orother sample, with a known amount or relative amount of HA. For example,a sample can include a tumor cell line known to express relatively lowlevels of HA. Exemplary of such tumor cell lines described herein thatexpress low levels of HA, for example, the HCT 116 cell line, the HT29cell line, the NCI H460 cell line, the DU145 cell line, the Capan-1 cellline, and tumors from tumor models generated using such cell lines.

It is understood that the particular change, e.g. increase in ordecrease in HA, is dependent on the assay used and the source of samplebeing measured. For example, in an ELISA, the fold increase or decreasein absorbance at a particular wavelength or in quantity of protein (e.g.as determined by using a standard curve) can be expressed relative to acontrol. In a PCR assay, such as RT-PCR, sample expression levels can becompared to control expression levels (e.g. expressed as fold change)using methods known to those in the art, such as using standards.

For example, when the amount of hyaluronan in a sample from a subject isbeing tested, detection of the marker can be determining that the amountof HA in the sample (e.g. cancerous cell, tissue or fluid) from thesubject is elevated compared to a control sample, such as a controlsample described in the previous paragraph. In one example, the canceris determined to be a hyaluronan-rich cancer if the amount of HA in thetissue, cell or fluid is elevated at or about 0.5-fold, 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, or more, comparedto the control sample.

In some examples, a tumor can be directly biopsied and stained forexpression of HA. In other examples, a sample, such as a blood or urinesample or other bodily fluid sample associated with the particular tumorcan be assayed for HA. The type of assay will vary depending on thetumor-type, although it is contemplated that more than one assay can beused to detect HA. References herein to such assays for particulartumors are for illustration only. For example, for bladder cancers,urine samples can be assayed for hyaluronan by standard ELISAprocedures. For purposes herein, subjects that exhibit 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or moreHA compared to urine from normal patient controls (see e.g., Lokeshwaret al. (2000) J. Urol., 163:348-56), can be selected. In anotherexample, tumor cells can be biopsied and stained for HA, such as byimmunohistochemistry (see e.g., Anttila et al. (2000) Cancer Research,60:150-156; Karvinen et al. (2003) British J of Dermatology, 148:86-94;Lipponen et al. (2001) Euro J Can. 37: 849-856); Auvinen et al. (2000)American J of Pathology, 156:529;). Generally, in such examples, a tumorsample or tumor cell is considered positive for HA if any cancer-cellassociated HA signal is observed. As a negative control for backgroundstaining, cells can be predigested with a hyaluronidase to cleave allcell-associated HA. Samples also can be compared to a normal cell ortissue from the same subject. In addition, in such methods, the level ofcell-associated hyaluronan can be scored as low, moderate or high. Forexample, HA expression is considered high or moderate if 30%, 35%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90% or more of the tumoral area showedpersistent HA signal. Typically, treatment of subjects with moderate tohigh HA is contemplated herein.

2. Use in Treating Cancers

As noted above, hyaluronan plays a role in processes associated withcancer and hyaluronan levels correlate with tumor aggressiveness, andvarious markers for tumor aggressiveness and poor prognosis. Thus,provided are methods for treating hyaluronan-associated cancers withanti-hyaluronan agents in combination with a corticosteroid toameliorate, reduce or lessen muscoloskeletal side effects induced by theenzyme. The cancers include hyaluronan-rich cancers and cancers that areassociated with elevated interstitial fluid pressure.

Hyaluronan plays a role in processes associated with cell motility,including development, regeneration, repair, embryogenesis,embryological development, wound healing, angiogenesis, andtumorigenesis (Toole 1991 Cell Biol. Extracell. Matrix, Hay (ed.),Plenum Press, New York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer52:1-6; Knudson et al, 1993 FASEB J. 7:1233-1241). In addition,hyaluronan levels correlate with tumor aggressiveness (Ozello et al.1960 Cancer Res. 20:600-604; Takeuchi et al. 1976, Cancer Res.36:2133-2139; Kimata et al. 1983 Cancer Res. 43:1347-1354); hyaluronanpromotes several cancer processes, including, but not limited to, tumorgrowth, survival, metastasis and interstitial fluid pressure.

Hyaluronidase has direct anticarcinogenic effects when injected intotumors. Hyaluronidase prevents growth of tumors transplanted into mice(De Maeyer et al., (1992) Int. J. Cancer 51:657-660) and inhibits tumorformation upon exposure to carcinogens (Pawlowski et al. (1979) Int. J.Cancer 23:105-109) Hyaluronidase is effective as the sole therapeuticagent in the treatment of brain cancer (gliomas) (WO 198802261). Asdiscussed above, other anti-hyaluronan agents also have known anti-tumoractivity.

Hyaluronan-associated cancers are cancers associated withhyaluronan-expression, typically elevated hyaluronan expression, whichcan be determined, for example, prior to treatment, as described above.Exemplary of the hyaluronan-associated diseases and conditions that canbe treated using the provided compositions containing an anti-hyaluronanagents and methods are cancers, particularly hyaluronan-rich cancers,for example, hyaluronan-rich cancers that are associated with elevatedinterstitial fluid pressure.

For example, the hyaluronan-rich cancer can be a cancer in which thecancer cells produce HALOs in an in vitro particle exclusion assay;cancers that have elevated expression of hyaluronan (as determined byimmunostaining, e.g. histological staining of sections from the tumor);cancers that have elevated HAS2 (Hyaluronan synthase 2); and cancersthat do not produce hyaluronidase (HYAL1) in vitro. Hyaluronan-richcancers can be identified by any method for assessing hyaluronanexpression as described herein, and other known methods for assayingprotein/mRNA expression.

Several hyaluronan-rich cancers have been identified. In some cases,hyaluronan expression correlates with poor prognosis, for example,decreased survival rate and/or recurrence-free survival rate,metastases, angiogenesis, cancer cell invasion into other tissues/areas,and other indicators of poor prognosis. Such correlation has beenobserved, for example, in hyaluronan-rich tumors including ovariancancer, SCC, ISC, prostate cancer, lung cancer, including non-small-celllung cancer (NSCLC), breast cancer, colon cancer and pancreatic cancer(see, for example, Maarit et al., Cancer Research, 60:150-155 (2000);Karvinen et al., British Journal of Dermatology, 148:86-94 (2003);Lipponen et al., Eur. Journal of Cancer, 849-856 (2001); Pirinen et al.,Int. J. Cancer: 95: 12-17 (2001); Auvinen et al., American Journal ofPathology, 156(2):529-536 (2000); Ropponen et al., Cancer Research, 58:342-347 (1998)). Thus, hyaluronan-rich cancers can be treated byadministration of an anti-hyaluronan agent, such as a hyaluronidase, totreat one or more symptoms of the cancer. Hyaluronan-rich tumorsinclude, but are not limited to, prostate, breast, colon, ovarian,stomach, head and neck and other tumors and cancers.

Anti-hyaluronan agents, such as hyaluronan degrading enzymes, includinghyaluronidases, can also be used to increase the sensitivity of tumorsthat are resistant to conventional chemotherapy. For example,anti-hyaluronan agents, for example a hyaluronan degrading enzymes,including hyaluronidases, such as rHuPH20, can be administered to apatient having a tumor associated with a HYAL1 defect in an amounteffective to increase diffusion around the tumor site (e.g., tofacilitate circulation and/or concentrations of chemotherapeutic agentsin and around the tumor site), inhibit tumor cell motility, such as byhyaluronic acid degradation, and/or to lower the tumor cell apoptosisthreshold. This can bring the tumor cell(s) to a state of anoikis, whichrenders the tumor cell more susceptible to the action ofchemotherapeutic agents. Administration of an anti-hyaluronan agent,such as a hyaluronidase, can induce responsiveness of previouslychemotherapy-resistant tumors of the pancreas, stomach, colon, ovaries,and breast (Baumgartner et al. (1988) Reg. Cancer Treat. 1:55-58; Zankeret al. (1986) Proc. Amer. Assoc. Cancer Res. 27:390).

In one example, anti-hyaluronan agents, such as hyaluronan degradingenzymes, in particular, hyaluronidases, are used in the treatment ofmetastatic and non-metastatic cancers, including those that havedecreased endogenous hyaluronidase activity relative to non-cancerouscells. Anti-hyaluronan agents, for example hyaluronan degrading enzymessuch as hyaluronidases, can be used as a chemotherapeutic agent alone orin combination with other chemotherapeutics. Exemplary cancers include,but are not limited to, small lung cell carcinoma, squamous lung cellcarcinoma, and cancers of the breast, ovaries, head and neck, or anyother cancer associated with depressed levels of hyaluronidase activityor decreased hyaluronic acid catabolism.

In addition to treatment of the disease with the anti-hyaluronan agentalone (and also in combination with a corticosteroid), the compositionsand methods provided herein also can be used to treathyaluronan-associated cancers by administration of the anti-hyaluronanagent in combination with, for example, simultaneously or prior to, achemotherapeutic or other anti-cancer agent or treatment. In thisexample, the anti-hyaluronan agent, for example a hyaluronan degradingenzyme, such as a hyaluronidase, typically enhances penetration ofchemotherapeutic or other anti-cancer agents into solid tumors, therebytreating the disease. The anti-hyaluronan agent, for example ahyaluronan degrading enzyme, such as a hyaluronidase, can be injectedintratumorally with anti-cancer agents or intravenously for disseminatedcancers or hard to reach tumors.

The anticancer agent can be a chemotherapeutic, an antibody, a peptide,or a gene therapy vector, virus or DNA. Additionally, an anti-hyaluronanagent, for example a hyaluronan degrading enzymes, such as ahyaluronidase, can be used to recruit tumor cells into the cycling poolfor sensitization in previously chemorefractory tumors that haveacquired multiple drug resistance (St Croix et al., (1998) Cancer LettSeptember 131(1): 35-44). Anti-hyaluronan agents, such as hyaluronandegrading enzymes, including hyaluronidases, such as, for example,rHuPH20, also can enhance delivery of biologics such as monoclonalantibodies, cytokines and other drugs to tumors that accumulateglycosaminoglycans.

Exemplary anti-cancer agents that can be administered after, coincidentwith or before administration of an anti-hyaluronan agent, for example ahyaluronan degrading enzyme, such as a hyaluronidase, include, but arenot limited to Acivicins; Aclarubicins; Acodazoles; Acronines;Adozelesins; Aldesleukins; Alemtuzumabs; Alitretinoins (9-Cis-RetinoicAcids); Allopurinols; Altretamines; Alvocidibs; Ambazones; Ambomycins;Ametantrones; Amifostines; Aminoglutethimides; Amsacrines; Anastrozoles;Anaxirones; Ancitabines; Anthramycins; Apaziquones; Argimesnas; ArsenicTrioxides; Asparaginases; Asperlins; Atrimustines; Azacitidines;Azetepas; Azotomycins; Banoxantrones; Batabulins; Batimastats; BCG Live;Benaxibines; Bendamustines; Benzodepas; Bexarotenes; Bevacizumab;Bicalutamides; Bietaserpines; Biricodars; Bisantrenes; Bisantrenes;Bisnafide Dimesylates; Bizelesins; Bleomycins; Bortezomibs; Brequinars;Bropirimines; Budotitanes; Busulfans; Cactinomycins; Calusterones;Canertinibs; Capecitabines; Caracemides; Carbetimers; Carboplatins;Carboquones; Carmofurs; Carmustines with Polifeprosans; Carmustines;Carubicins; Carzelesins; Cedefingols; Celecoxibs; Cemadotins;Chlorambucils; Cioteronels; Cirolemycins; Cisplatins; Cladribines;Clanfenurs; Clofarabines; Crisnatols; Cyclophosphamides; Cytarabineliposomals; Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;Decitabines; Denileukin Diftitoxes; Dexniguldipines; Dexonnaplatins;Dexrazoxanes; Dezaguanines; Diaziquones; Dibrospidiums; Dienogests;Dinalins; Disermolides; Docetaxels; Dofequidars; Doxifluridines;Doxorubicin liposomals; Doxorubicin HCL; Doxorubicin HCL liposomeinjection; Doxorubicins; Droloxifenes; Dromostanolone Propionates;Duazomycins; Ecomustines; Edatrexates; Edotecarins; Eflornithines;Elacridars; Elinafides; Elliott's B Solutions; Elsamitrucins; Emitefurs;Enloplatins; Enpromates; Enzastaurins; Epipropidines; Epirubicins;Epoetin alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles; Fazarabines;Fenretinides; Filgrastims; Floxuridines; Fludarabines; Fluorouracils;5-fluorouracils; Fluoxymesterones; Fluorocitabines; Fosquidones;Fostriecins; Fostriecins; Fotretamines; Fulvestrants; Galarubicins;Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins; Geroquinols;Gimatecans; Gimeracils; Gloxazones; Glufosfamides; Goserelin acetates;Hydroxyureas; Ibritumomabs/Tiuxetans; Idarubicins; Ifosfamides;Ilmofosines; Ilomastats; Imatinib mesylates; Imexons; Improsulfans;Indisulams; Inproquones; Interferon alfa-2 as; Interferon alfa-2bs;Interferon Alfas; Interferon Betas; Interferon Gammas; Interferons;Interleukin-2s and other Interleukins (including recombinantInterleukins); Intoplicines; Iobenguanes [131-I]; Iproplatins;Irinotecans; Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines;Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins;Leuprolides; Leuprorelins (Leuprorelides); Levamisoles; Lexacalcitols;Liarozoles; Lobaplatins; Lometrexols; Lomustines/CCNUs; Lomustines;Lonafarnibs; Losoxantrones; Lurtotecans; Mafosfamides; Mannosulfans;Marimastats; Masoprocols; Maytansines; Mechlorethamines;Mechlorethamines/Nitrogen mustards; Megestrol acetates; Megestrols;Melengestrols; Melphalans; MelphalanslL-PAMs; Menogarils; Mepitiostanes;Mercaptopurines; 6-Mercaptopurine; Mesnas; Metesinds; Methotrexates;Methoxsalens; Metomidates; Metoprines; Meturedepas; Miboplatins;Miproxifenes; Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone Phenpropionates;Nedaplatins; Nelzarabines; Nemorubicins; Nitracrines; Nocodazoles;Nofetumomabs; Nogalamycins; Nolatrexeds; Nortopixantrones; Octreotides;Oprelvekins; Ormaplatins; Ortataxels; Oteracils; Oxaliplatins;Oxisurans; Oxophenarsines; Paclitaxels; Pamidronates; Patubilones;Pegademases; Pegaspargases; Pegfilgrastims; Peldesines; Peliomycins;Pelitrexols; Pemetrexeds; Pentamustines; Pentostatins; Peplomycins;Perfosfamides; Perifosines; Picoplatins; Pinafides; Pipobromans;Piposulfans; Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds;Plicamycid Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimersodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins; Sabarubicins;Safingols; Sargramostims; Satraplatins; Sebriplatins; Semustines;Simtrazenes; Sizofurans; Sobuzoxanes; Sorafenibs; Sparfosates; SparfosicAcids; Sparsomycins; Spirogermaniums; Spiromustines; Spiroplatins;Spiroplatins; Squalamines; Streptonigrins; Streptovarycins;Streptozocins; Sufosfamides; Sulofenurs; Sunitinib Malate; 6-thioguanine(6-TG); Tacedinalines; Talcs; Talisomycins; Tallimustines; Tamoxifens;Tariquidars; Tauromustines; Tecogalans; Tegafurs; Teloxantrones;Temoporfins; Temozolomides; Teniposides/VM-26s; Teniposides;Teroxirones; Testolactones; Thiamiprines; Thioguanines; Thiotepas;Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;Tirapazamines; Topixantrones; Topotecans; Toremifenes; Tositumomabs;Trabectedins (Ecteinascidin 743); Trastuzumabs; Trestolones;Tretinoins/ATRA; Triciribines; Trilostanes; Trimetrexates; TriplatinTetranitrates; Triptorelins; Trofosfamides; Tubulozoles; Ubenimexs;Uracil Mustards; Uredepas; Valrubicins; Valspodars; Vapreotides;Verteporfins; Vinblastines; Vincristines; Vindesines; Vinepidines;Vinflunines; Vinformides; Vinglycinates; Vinleucinols; Vinleurosines;Vinorelbines; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles;Xanthomycin As (Guamecyclines); Zeniplatins; Zilascorbs [2-H];Zinostatins; Zoledronate; Zorubicins; and Zosuquidars, for example:

Aldesleukins (e.g. PROLEUKIN®); Alemtuzumabs (e.g. CAMPATH®);Alitretinoins (e.g. PANRETIN®); Allopurinols (e.g. ZYLOPRIM®);Altretamines (e.g. HEXALEN®); Amifostines (e.g. ETHYOL®); Anastrozoles(e.g. ARIMIDEX®); Arsenic Trioxides (e.g. TRISENOX®); Asparaginases(e.g. ELSPAR®); BCG Live (e.g. TICE® BCG); Bexarotenes (e.g.TARGRETIN®); Bevacizumab (AVASTIN®); Bleomycins (e.g. BLENOXANE®);Busulfan intravenous (e.g. BUSULFEX®); Busulfan orals (e.g. MYLERAN®);Calusterones (e.g. METHOSARB®); Capecitabines (e.g. XELODA®);Carboplatins (e.g. PARAPLATIN®); Carmustines (e.g. BCNU®, BiCNU®);Carmustines with Polifeprosans (e.g. GLIADEL® Wafer); Celecoxibs (e.g.CELEBREX®); Chlorambucils (e.g. LEUKERAN®); Cisplatins (e.g. PLATINOL®);Cladribines (e.g. LEUSTATIN®, 2-CdA®); Cyclophosphamides (e.g. CYTOXAN®,NEOSAR®); Cytarabines (e.g. CYTOSAR-U®); Cytarabine liposomals (e.g.DepoCyt®); Dacarbazines (e.g. DTIC-Dome): Dactinomycins (e.g.COSMEGEN®); Darbepoetin Alfas (e.g. ARANESP®); Daunorubicin liposomals(e.g. DANUOXOME®); Daunorubicins/Daunomycins (e.g. CERUBIDINE®);Denileukin Diftitoxes (e.g. ONTAK®); Dexrazoxanes (e.g. ZINECARD®);Docetaxels (e.g. TAXOTERE®); Doxorubicins (e.g. ADRIAMYCIN®, RUBEX®);Doxorubicin liposomals, including Doxorubicin HCL liposome injections(e.g. DOXIL®); Dromostanolone propionates (e.g. DROMOSTANOLONE® andMASTERONE® Injection); Elliott's B Solutions (e.g. Elliott's BSolution®); Epirubicins (e.g. ELLENCE®); Epoetin alfas (e.g. EPOGEN®);Estramustines (e.g. EMCYT®); Etoposide phosphates (e.g. ETOPOPHOS®);Etoposide VP-16s (e.g. VEPESID®); Exemestanes (e.g. AROMASIN®);Filgrastims (e.g. NEUPOGEN®); Floxuridines (e.g. FUDR®); Fludarabines(e.g. FLUDARA®); Fluorouracils incl. 5-FUs (e.g. ADRUCIL®); Fulvestrants(e.g. FASLODEX®); Gemcitabines (e.g. GEMZAR®); Gemtuzumabs/Ozogamicins(e.g. MYLOTARG®); Goserelin acetates (e.g. ZOLADEX®); Hydroxyureas (e.g.HYDREA®); Ibritumomabs/Tiuxetans (e.g. ZEVALIN®); Idarubicins (e.g.IDAMYCIN®); Ifosfamides (e.g. IFEX®); Imatinib mesylates (e.g.GLEEVEC®); Interferon alfa-2 as (e.g. ROFERON-A®); Interferon alfa-2bs(e.g. INTRON A®); Irinotecans (e.g. CAMPTOSAR®); Letrozoles (e.g.FEMARA®); Leucovorins (e.g. WELLCOVORIN®, LEUCOVORIN®); Levamisoles(e.g. ERGAMISOL®); Lomustines/CCNUs (e.g. CeeBU®);Mechlorethamines/Nitrogen mustards (e.g. MUSTARGEN®); Megestrol acetates(e.g. MEGACE®); Melphalans/L-PAMs (e.g. ALKERAN®); Mercaptopurine,including 6-mercaptopurines (6-MPs; e.g. PURINETHOL®); Mesnas (e.g.MESNEX®); Methotrexates; Methoxsalens (e.g. UVADEX®); Mitomycin Cs (e.g.MUTAMYCIN®, MITOZYTREX®); Mitotanes (e.g. LYSODREN®); Mitoxantrones(e.g. NOVANTRONE®); Nandrolone Phenpropionates (e.g. DURABOLIN-50®);Nofetumomabs (e.g. VERLUMA®); Oprelvekins (e.g. NEUMEGA®); Oxaliplatins(e.g. ELOXATIN®); Paclitaxels (e.g. PAXENE®, TAXOL®); Pamidronates (e.g.AREDIA®); Pegademases (e.g. ADAGEN®); Pegaspargases (e.g. ONCASPAR®);Pegfilgrastims (e.g. NEULASTA®); Pentostatins (e.g. NIPENT®);Pipobromans (e.g. VERCYTE®); Plicamycin/Mithramycins (e.g. MITHRACIN®);Porfimer sodiums (e.g. PHOTOFRIN®); Procarbazines (e.g. MATULANE®);Quinacrines (e.g. ATABRINE®); Rasburicases (e.g. ELITEK®); Rituximabs(e.g. RITUXAN®); Sargramostims (e.g. PROKINE®); Streptozocins (e.g.ZANOSAR®); Sunitinib Malates (e.g. SUTENT®); Talcs (e.g. SCLEROSOL®);Tamoxifens (e.g. NOLVADEX®); Temozolomides (e.g. TEMODAR®);Teniposides/VM-26s (e.g. VUMON®); Testolactones (e.g. TESLAC®);Thioguanines including, 6-thioguanine (6-TG); Thiotepas (e.g.THIOPLEX®); Topotecans (e.g. HYCAMTIN®); Toremifenes (e.g. FARESTON®);Tositumomabs (e.g. BEXXAR®); Trastuzumabs (e.g. HERCEPTIN®);Tretinoins/ATRA (e.g. VESANOID®); Uracil Mustards; Valrubicins (e.g.VALSTAR®); Vinblastines (e.g. VELBAN®); Vincristines (e.g. ONCOVIN®);Vinorelbines (e.g. NAVELBINE®); and Zoledronates (e.g. ZOMETA®).

In one example, an anti-hyaluronan agent, for example a hyaluronandegrading enzyme, such as a modified hyaluronidase, for example,PEGylated rHuPH20, is administered to a subject after, coincident withor before administration of one or more of docetaxel (e.g. TAXOTERE®),Doxorubicin liposomal (e.g. DOXIL®), Sunitinib Malate (e.g. SUTENT®) orBevacizumab (AVASTIN®). In the methods herein, a corticosteroid also isadministered to ameliorate or prevent side effects, such asmusculoskeletal side effects resulting from or associated withadministration of the anti-hyaluronan agent.

I. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 rHuPH20 Expressing Cell Lines

A. Generation of an Initial Soluble rHuPH20-Expressing Cell Line

Chinese Hamster Ovary (CHO) cells were transfected with the HZ24 plasmid(set forth in SEQ ID NO:52). The HZ24 plasmid vector for expression ofsoluble rHuPH20 contains a pCI vector backbone (Promega), DNA encodingamino acids 1-482 of human PH20 hyaluronidase (SEQ ID NO:49), aninternal ribosomal entry site (IRES) from the ECMV virus (Clontech), andthe mouse dihydrofolate reductase (DHFR) gene. The pCI vector backbonealso includes DNA encoding the Beta-lactamase resistance gene (AmpR), anf1 origin of replication, a Cytomegalovirus immediate-earlyenhancer/promoter region (CMV), a chimeric intron, and an SV40 latepolyadenylation signal (SV40). The DNA encoding the soluble rHuPH20construct contains an NheI site and a Kozak consensus sequence prior tothe DNA encoding the methionine at amino acid position 1 of the native35 amino acid signal sequence of human PH20, and a stop codon followingthe DNA encoding the tyrosine corresponding to amino acid position 482of the human PH20 hyaluronidase set forth in SEQ ID NO:1), followed by aBamHI restriction site. The construct pCI-PH20-IRES-DHFR-SV40pa (HZ24),therefore, results in a single mRNA species driven by the CMV promoterthat encodes amino acids 1-482 of human PH20 (set forth in SEQ ID NO:3)and amino acids 1-186 of mouse dihydrofolate reductase (set forth in SEQID NO:53), separated by the internal ribosomal entry site (IRES).

Non-transfected CHO cells growing in GIBCO Modified CD-CHO media forDHFR(−) cells, supplemented with 4 mM Glutamine and 18 ml/L PlurionicF68/L (Gibco), were seeded at 0.5×10⁶ cells/ml in a shaker flask inpreparation for transfection. Cells were grown at 37° C. in 5% CO₂ in ahumidified incubator, shaking at 120 rpm. Exponentially growingnon-transfected CHO cells were tested for viability prior totransfection.

Sixty million viable cells of the non-transfected CHO cell culture werepelleted and resuspended to a density of 2×10⁷ cells in 0.7 mL of 2×transfection buffer (2×HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mMKCl, 1.4 mM Na₂HPO₄, 12 mM dextrose). To each aliquot of resuspendedcells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized byovernight digestion with Cla I (New England Biolabs) was added, and thecell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics)electroporation cuvettes at room temperature. A negative controlelectroporation was performed with no plasmid DNA mixed with the cells.The cell/plasmid mixes were electroporated with a capacitor discharge of330 V and 960 μF or at 350 V and 960 μF.

The cells were removed from the cuvettes after electroporation andtransferred into 5 mL of Modified CD-CHO media for DHFR(−) cells,supplemented with 4 mM Glutamine and 18 ml/L Plurionic F68/L (Gibco),and allowed to grow in a well of a 6-well tissue culture plate withoutselection for 2 days at 37° C. in 5% CO₂ in a humidified incubator.

Two days post-electroporation, 0.5 mL of tissue culture media wasremoved from each well and tested for the presence of hyaluronidaseactivity using the microturbidity assay described in Example 4. Cellsexpressing the highest levels of hyaluronidase activity were collectedfrom the tissue culture well, counted and diluted to 1×10⁴ to 2×10⁴viable cells per mL. A 0.1 mL aliquot of the cell suspension wastransferred to each well of five, 96 well round bottom tissue cultureplates. One hundred microliters of CD-CHO media (GIBCO) containing 4 mMGlutaMAX™-1 supplement (GIBCO™, Invitrogen Corporation) and withouthypoxanthine and thymidine supplements were added to the wellscontaining cells (final volume 0.2 mL).

Ten clones were identified from the 5 plates grown without methotrexate.Six of these HZ24 clones were expanded in culture and transferred intoshaker flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9,1E11, and 4D10 were plated into 96-well round bottom tissue cultureplates using a two-dimensional infinite dilution strategy in which cellswere diluted 1:2 down the plate, and 1:3 across the plate, starting at5000 cells in the top left hand well. Diluted clones were grown in abackground of 500 non-transfected DG44 CHO cells per well, to providenecessary growth factors for the initial days in culture. Ten plateswere made per subclone, with 5 plates containing 50 nM methotrexate and5 plates without methotrexate.

Clone 3D3 produced 24 visual subclones (13 from the no methotrexatetreatment, and 11 from the 50 nM methotrexate treatment). Significanthyaluronidase activity was measured in the supernatants from 8 of the 24subclones (>50 Units/mL), and these 8 subclones were expanded into T-25tissue culture flasks. Clones isolated from the methotrexate treatmentprotocol were expanded in the presence of 50 nM methotrexate. Clone3D35M was further expanded in 500 nM methotrexate in shaker flasks andgave rise to clones producing in excess of 1,000 Units/ml hyaluronidaseactivity (clone 3D35M; or Gen1 3D35M). A master cell bank (MCB) of the3D35M cells was then prepared

B. Generation of a Second Generation Cell Line Expressing SolublerHuPH20

The Gen1 3D35M cell line described in Example 1A was adapted to highermethotrexate levels to produce generation 2 (Gen2) clones. 3D35M cellswere seeded from established methotrexate-containing cultures into CDCHO medium containing 4 mM GlutaMAX-1™ and 1.0 μM methotrexate. Thecells were adapted to a higher methotrexate level by growing andpassaging them 9 times over a period of 46 days in a 37° C., 7% CO₂humidified incubator. The amplified population of cells was cloned outby limiting dilution in 96-well tissue culture plates containing mediumwith 2.0 μM methotrexate. After approximately 4 weeks, clones wereidentified and clone 3E10B was selected for expansion. 3E10B cells weregrown in CD CHO medium containing 4 mM GlutaMAX-1™ and 2.0 μMmethotrexate for 20 passages. A master cell bank (MCB) of the 3E10B cellline was created and frozen and used for subsequent studies.

Amplification of the cell line continued by culturing 3E10B cells in CDCHO medium containing 4 mM GlutaMAX-1™ and 4.0 μM methotrexate. Afterthe 12^(th) passage, cells were frozen in vials as a research cell bank(RCB). One vial of the RCB was thawed and cultured in medium containing8.0 μM methotrexate. After 5 days, the methotrexate concentration in themedium was increased to 16.0 μM, then 20.0 μM 18 days later. Cells fromthe 8^(th) passage in medium containing 20.0 μM methotrexate were clonedout by limiting dilution in 96-well tissue culture plates containing CDCHO medium containing 4 mM GlutaMAX-1™ and 20.0 μM methotrexate. Cloneswere identified 5-6 weeks later and clone 2B2 was selected for expansionin medium containing 20.0 μM methotrexate. After the 11th passage, 2B2cells were frozen in vials as a research cell bank (RCB).

The resultant 2B2 cells are dihydrofolate reductase deficient (dhfr−)DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20).The soluble PH20 is present in 2B2 cells at a copy number ofapproximately 206 copies/cell. Southern blot analysis of Spe I-, Xba I-and BamH I/Hind III-digested genomic 2B2 cell DNA using arHuPH20-specific probe revealed the following restriction digestprofile: one major hybridizing band of ˜7.7 kb and four minorhybridizing bands (˜13.9, ˜6.6, ˜5.7 and ˜4.6 kb) with DNA digested withSpe I; one major hybridizing band of ˜5.0 kb and two minor hybridizingbands (˜13.9 and ˜6.5 kb) with DNA digested with Xba I; and one singlehybridizing band of ˜1.4 kb observed using 2B2 DNA digested with BamHI/Hind III. Sequence analysis of the mRNA transcript indicated that thederived cDNA (SEQ ID NO:56) was identical to the reference sequence (SEQID NO:49) except for one base pair difference at position 1131, whichwas observed to be a thymidine (T) instead of the expected cytosine (C).This is a silent mutation, with no effect on the amino acid sequence.

Example 2 Production and Purification of rHuPH20

A. Production of Gen2 soluble rHuPH20 in 300 L Bioreactor Cell Culture

A vial of HZ24-2B2 cells (Example 1B) was thawed and expanded fromshaker flasks through 36 L spinner flasks in CD-CHO media (Invitrogen,Carlsbad, Calif.) supplemented with 20 μM methotrexate and GlutaMAX-1™(Invitrogen). Briefly, a vial of cells was thawed in a 37° C. waterbath, media was added and the cells were centrifuged. The cells werere-suspended in a 125 mL shake flask with 20 mL of fresh media andplaced in a 37° C., 7% CO₂ incubator. The cells were expanded up to 40mL in the 125 mL shake flask. When the cell density reached greater than1.5×10⁶ cells/mL, the culture was expanded into a 125 mL spinner flaskin a 100 mL culture volume. The flask was incubated at 37° C., 7% CO₂.When the cell density reached greater than 1.5×10⁶ cells/mL, the culturewas expanded into a 250 mL spinner flask in 200 mL culture volume, andthe flask was incubated at 37° C., 7% CO₂. When the cell density reachedgreater than 1.5×10⁶ cells/mL, the culture was expanded into a 1 Lspinner flask in 800 mL culture volume and incubated at 37° C., 7% CO₂.When the cell density reached greater than 1.5×10⁶ cells/mL the culturewas expanded into a 6 L spinner flask in 5000 mL culture volume andincubated at 37° C., 7% CO₂. When the cell density reached greater than1.5×10⁶ cells/mL the culture was expanded into a 36 L spinner flask in32 L culture volume and incubated at 37° C., 7% CO₂.

A 400 L reactor was sterilized and 230 mL of CD-CHO media was added.Before use, the reactor was checked for contamination. Approximately 30L cells were transferred from the 36 L spinner flasks to the 400 Lbioreactor (Braun) at an inoculation density of 4.0×10⁵ viable cells perml and a total volume of 260 L. Parameters were temperature set point,37° C.; Impeller Speed 40-55 RPM; Vessel Pressure: 3 psi; Air Sparge0.5-1.5 L/Min.; Air Overlay: 3 L/min. The reactor was sampled daily forcell counts, pH verification, media analysis, protein production andretention. Also, during the run nutrient feeds were added. At 120 hrs(day 5), 10.4 L of Feed #1 Medium (4×CD-CHO+33 g/L Glucose+160 mL/LGlutamax-1™+83 mL/L Yeastolate+33 mg/L rHuInsulin) was added. At 168hours (day 7), 10.8 L of Feed #2 (2×CD-CHO+33 g/L Glucose+80 mL/LGlutamax-1™+167 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was added, andculture temperature was changed to 36.5° C. At 216 hours (day 9), 10.8 Lof Feed #3 (1×CD-CHO+50 g/L Glucose+50 mL/L Glutamax-1™+250 mL/LYeastolate+1.80 g/L Sodium Butyrate) was added, and culture temperaturewas changed to 36° C. At 264 hours (day 11), 10.8 L of Feed #4(1×CD-CHO+33 g/L Glucose+33 mL/L Glutamax-1™+250 mL/L Yeastolate+0.92g/L Sodium Butyrate) was added, and culture temperature was changed to35.5° C. The addition of the feed media was observed to dramaticallyenhance the production of soluble rHuPH20 in the final stages ofproduction. The reactor was harvested at 14 or 15 days or when theviability of the cells dropped below 40%. The process resulted in afinal productivity of 17,000 Units per ml with a maximal cell density of12 million cells/mL. At harvest, the culture was sampled for mycoplasma,bioburden, endotoxin and viral in vitro and in vivo, TransmissionElectron Microscopy (TEM) and enzyme activity.

The culture was pumped by a peristaltic pump through four Millistakfiltration system modules (Millipore) in parallel, each containing alayer of diatomaceous earth graded to 4-8 μm and a layer of diatomaceousearth graded to 1.4-1.1 μm, followed by a cellulose membrane, thenthrough a second single Millistak filtration system (Millipore)containing a layer of diatomaceous earth graded to 0.4-0.11 μm and alayer of diatomaceous earth graded to <0.1 μm, followed by a cellulosemembrane, and then through a 0.22 μm final filter into a sterile singleuse flexible bag with a 350 L capacity. The harvested cell culture fluidwas supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. Theculture was concentrated 10× with a tangential flow filtration (TFF)apparatus using four Sartoslice TFF 30 kDa molecular weight cut-off(MWCO) polyether sulfone (PES) filters (Sartorius), followed by a 10×buffer exchange with 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 into a 0.22 μmfinal filter into a 50 L sterile storage bag.

The concentrated, diafiltered harvest was inactivated for virus. Priorto viral inactivation, a solution of 10% Triton X-100, 3% tri (n-butyl)phosphate (TNBP) was prepared. The concentrated, diafiltered harvest wasexposed to 1% Triton X-100, 0.3% TNBP for 1 hour in a 36 L glassreaction vessel immediately prior to purification on the Q column.

B. Purification of Gen2 Soluble rHuPH20

A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H=29 cm, D=20cm) was prepared. Wash samples were collected for a determination of pH,conductivity and endotoxin (LAL) assay. The column was equilibrated with5 column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5. Following viralinactivation, the concentrated, diafiltered harvest (Example 2A) wasloaded onto the Q column at a flow rate of 100 cm/hr. The column waswashed with 5 column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5 and 10mM Hepes, 50 mM NaCl, pH 7.0. The protein was eluted with 10 mM Hepes,400 mM NaCl, pH 7.0 into a 0.22 μm final filter into a sterile bag. Theeluate sample was tested for bioburden, protein concentration andhyaluronidase activity. A₂₈₀ absorbance readings were taken at thebeginning and end of the exchange.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm,D=30 cm) was prepared. The wash was collected and sampled for pH,conductivity and endotoxin (LAL assay). The column was equilibrated with5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate,0.1 mM CaCl₂, pH 7.0. The protein eluate from the Q sepharose column wassupplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1 MCaCl₂ stock solutions to yield final concentrations of 5 mM, 0.5 M and0.1 mM, respectively. The protein was loaded onto the PS column at aflow rate of 100 cm/hr and the column flow thru collected. The columnwas washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1mM CaCl₂ pH 7.0 at 100 cm/hr and the wash was added to the collectedflow thru. Combined with the column wash, the flow through was passedthrough a 0.22 μm final filter into a sterile bag. The flow through wassampled for bioburden, protein concentration and enzyme activity.

An aminophenyl boronate column (ProMedics) was prepared. The wash wascollected and sampled for pH, conductivity and endotoxin (LAL assay).The column was equilibrated with 5 column volumes of 5 mM potassiumphosphate, 0.5 M ammonium sulfate. The PS flow through containingpurified protein was loaded onto the aminophenyl boronate column at aflow rate of 100 cm/hr. The column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with50 mM Hepes, 100 mM NaCl, pH 6.9 and passed through a sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The hydroxyapatite (HAP) column (Biorad) was prepared. The wash wascollected and tested for pH, conductivity and endotoxin (LAL assay). Thecolumn was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1mM CaCl₂, pH 7.0. The aminophenyl boronate purified protein wassupplemented to final concentrations of 5 mM potassium phosphate and 0.1mM CaCl₂ and loaded onto the HAP column at a flow rate of 100 cm/hr. Thecolumn was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1mM CaCl₂. The column was next washed with 10 mM potassium phosphate, pH7, 100 mM NaCl, 0.1 mM CaCl₂. The protein was eluted with 70 mMpotassium phosphate, pH 7.0 and passed through a 0.22 μm sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The HAP purified protein was then passed through a viral removal filter.The sterilized Viosart filter (Sartorius) was first prepared by washingwith 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filteredbuffer was sampled for pH and conductivity. The HAP purified protein waspumped via a peristaltic pump through the 20 nM viral removal filter.The filtered protein in 70 mM potassium phosphate, pH 7.0 was passedthrough a 0.22 μM final filter into a sterile bag. The viral filteredsample was tested for protein concentration, enzyme activity,oligosaccharide, monosaccharide and sialic acid profiling. The samplealso was tested for process related impurities.

Example 3 Preparation of PEGylated rHuPH20

In this example, rHuPH20 was PEGylated by reaction of the enzyme withlinear N-hydroxysuccinimidyl ester of methoxy poly(ethylene glycol)butanoic acid (mPEG-SBA-30K).

A. Preparation of mPEG-SBA-30K

In order to generate PEGPH20, rHuPH20 (which is approximately 60 KDa insize) was covalently conjugated to a linear N-hydroxysuccinimidyl esterof methoxy poly(ethylene glycol) butanoic acid (mPEG-SBA-30K), having anapproximate molecular weight of 30 kDa. The structure of mPEG-SBA isshown below, where n 681.

Methods used to prepare the mPEG-SBA-30K that was used to PEGylaterHuPH20 are described, for example, in U.S. Pat. No. 5,672,662. Briefly,the mPEG-SBA-30K is made according to the following procedure:

A solution of ethyl malonate (2 equivalents) dissolved in dioxane isadded drop by drop to sodium hydride (2 equivalents) and toluene under anitrogen atmosphere. mPEG methane sulfonate (1 equivalent, MW 30 kDa,Shearwater) is dissolved in toluene and added to the above mixture. Theresulting mixture is refluxed for approximately 18 hours. The reactionmixture is concentrated to half its original volume, extracted with 10%aqueous NaCl solution, extracted with 1% aqueous hydrochloric acid, andthe aqueous extracts are combined. The collected aqueous layers areextracted with dichloromethane (3×) and the organic layer is dried withmagnesium sulfate, filtered and evaporated to dryness. The resultingresidue is dissolved in 1N sodium hydroxide containing sodium chlorideand the mixture is stirred for 1 hour. The pH of the mixture is adjustedto approximately 3 by addition of 6N hydrochloric acid. The mixture isextracted with dichloromethane (2×).

The organic layer is dried over magnesium sulfate, filtered,concentrated, and poured into cold diethyl ether. The precipitate iscollected by filtration and dried under vacuum. The resulting compoundis dissolved in dioxane and refluxed for 8 hours and then concentratedto dryness. The resulting residue is dissolved in water and extractedwith dichloromethane (2×), dried over magnesium sulfate, and thesolution is concentrated by rotary evaporation and then poured into colddiethyl ether. The precipitate is collected by filtration and driedunder vacuum. The resulting compound (1 equivalent) is dissolved indichloromethane and N-hydroxysuccinimide (2.1 equivalents) is added. Thesolution is cooled to 0° C. and a solution of dicyclohexylcarbodiimide(2.1 equivalents) in dichloromethane is added dropwise. The solution isstirred at room temperature for approximately 18 hours. The reactionmixture is filtered, concentrated and precipitated in diethyl ether. Theprecipitate is collected by filtration and dried under vacuum to affordthe powder mPEG-SBA-30K which is then frozen at ≦−15° C.

B. Conjugation of mPEG-SBA-30K to rHuPH20

To make the PEGPH20, mPEG-SBA-30K was coupled to the amino group(s) ofrHuPH20 by covalent conjugation, providing stable amide bonds betweenrHuPH20 and mPEG, as shown below, where n≈681.

Prior to congugation, the rHuPH20 purified bulk protein made in Example2B was concentrated to 10 mg/mL, using a 10 kDa polyethersulfone (PES)tangential flow filtration (TFF) cassettes (Sartorius) with a 0.2 m²filtration area, and buffer exchanged against 70 mM Potassium Phosphateat pH 7.2. The concentrated protein was then stored at 2-8° C. untiluse.

To conjugate the rHuPH20, the mPEG-SBA-30K (Nektar) was thawed at roomtemperature in the dark for not longer than 2 hours. Depending on thebatch size, a sterile 3″ stir bar was placed into a 1 or 3 literErlenmeyer flask and buffer exchanged rHuPH20 protein was added. Fivegrams of dry mPEG-SBA-30K powder per gram of rHuPH20 (10:1 molar ratioof mPEG-SBA-30K:rHuPH20) was added to the flask under a vacuum hood andthe mixture was mixed for 10 minutes or until the mPEG-SBA-30K wascomplete dissolved. The stir rate was set such that vortexing occurredwithout foaming.

The solution was then filtered under a class 100 hood by pumping thesolution, via peristaltic pump, through a 0.22 μm polystyrene, celluloseacetate filter capsule (Corning 50 mL Tubetop filter) into a new 1 or 3liter Erlenmeyer flask containing a sterile 3″ stir bar. The volume ofthe PEGPH20 reaction mixture was determined by mass (1 g/mL density) andthe 0.22 μm filter used for filtration was examined in a post-useintegrity test.

The mixture was then placed on a stir plate at 2-8° C. and mixed for20±1 hours in the dark. The stir rate was again set such that vortexingoccurred without foaming. The entire Erlenmeyer container was wrapped infoil to protect the solution from light. After mixing, the reaction wasquenched by adding 1M glycine to a final concentration of 25 mM. Sampleswere removed from the container to test pH and conductivity. The pH andconductivity were then adjusted by adding to a solution of mM Tris Base(5.65 L/L) and 5 mM Tris, 10 mM NaCl, pH 8.0 (13.35 L/L) to proceed withQ Sepharose purification.

A QFF Sepharose (GE Healthcare) ion exchange column (Height=21.5-24.0cm, Diameter=20 cm) was prepared by equilibration with 5 column volumes(36 L) of 5 mM Tris, 10 mM NaCl, pH 8.0. The congugated product wasloaded onto the QFF column at a flow rate of 95 cm/hr. The column wasthen washed with 11 L of equilibration buffer (5 mM Tris, 10 mM NaCl, pH8.0) at a flow rate of 95 cm/hr followed by a wash with 25 L ofequilibration buffer at a flow rate of 268 cm/hr. The protein productwas then eluted with 5 mM Tris, 130 mM NaCl, pH 8.0 at a flow rate of268 cm/hr. The resulting purified PEGPH20 was concentrated to 3.5 mg/mL,using a 30 lcDa polyethersulfone (PES) tangential flow filtration (TFF)cassettes (Sartorius) with a 0.2 m² filtration area, and bufferexchanged against 10 mM Histidine, 130 mM NaCl at pH 6.5. The resultingmaterial was tested for enzyme activity as described in Example 4 below.The PEGylated rHuPH20 material at a concentration of 3.5 mg/mL (finalenzyme activity 140,000 U/mL) was filled, in 3 mL volumes, into 5 mLglass vials with a siliconized bromobutyl rubber stopper and aluminumflip-off seal, and frozen (frozen overnight in a −20° C. freezer, thenput in a −80° C. freezer for longer storage). The PEGylated rHuHP20contained approximately 4.5 moles of PEG per mole of rHuPH20.

Example 4 Determination of Hyaluronidase Activity of Soluble rHuPH20

Hyaluronidase activity of soluble rHuPH20 in samples such as cellcultures, plasma, purification fractions and purified solutions wasdetermined using either a turbidimetric assay, which is based on theformation of an insoluble precipitate when hyaluronic acid binds withserum albumin, or a biotinylated-hyaluronic acid substrate assay, whichmeasures the amount of enzymatically active rHuPH20 or PEGPH20 by thedigestion of biotinylated hyaluronic acid (b-HA) substratenon-covalently bound to plastic multi-well microtiter plates.

A. Microturbidity Assay

Hyaluronidase activity of soluble rHuPH20 is measured by incubatingsoluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a setperiod of time (10 minutes) and then precipitating the undigested sodiumhyaluronate with the addition of acidified serum albumin. The turbidityof the resulting sample is measured at 640 nm after a 30 minutedevelopment period. The decrease in turbidity resulting from enzymeactivity on the sodium hyaluronate substrate is a measure of the solublerHuPH20 hyaluronidase activity. The method is performed using acalibration curve generated with dilutions of a soluble rHuPH20 assayworking reference standard, and sample activity measurements are maderelative to this calibration curve.

Dilutions of the sample were prepared in Enzyme Diluent Solutions. TheEnzyme Diluent Solution was prepared by dissolving 33.0±0.05 mg ofhydrolyzed gelatin in 25.0 mL of the 50 mM PIPES Reaction Buffer (140 mMNaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of sterile water for injection(SWFI), and diluting 0.2 mL of 25% Buminate solution into the mixtureand vortexing for 30 seconds. This was performed within 2 hours of useand stored on ice until needed. The samples were diluted to an estimated1-2 U/mL. Generally, the maximum dilution per step did not exceed 1:100and the initial sample size for the first dilution was not less than 20μL. The minimum sample volumes needed to perform the assay were asfollows: In-process Samples, FPLC Fractions: 80 μL; Tissue CultureSupernatants: 1 mL; Concentrated Material: 80 μL; Purified or Final StepMaterial: 80 μL. The dilutions were made in triplicate in a Low ProteinBinding 96-well plate, and 30 μL of each dilution was transferred toOptilux black/clear bottom plates (BD BioSciences).

Dilutions of known soluble rHuPH20 with a concentration of 2.5 U/mL wereprepared in Enzyme Diluent Solution to generate a standard curve andadded to the Optilux plate in triplicate. The dilutions included 0 U/mL,0.25 U/mL, 0.5 U/mL, 1.0 U/mL, 1.5 U/mL, 2.0 U/mL, and 2.5 U/mL.“Reagent blank” wells that contained 60 μL of Enzyme Diluent Solutionwere included in the plate as a negative control. The plate was thencovered and warmed on a heat block for 5 minutes at 37° C. The cover wasremoved and the plate was shaken for 10 seconds. After shaking, theplate was returned to the heat block and the MULTIDROP 384 LiquidHandling Device was primed with the warm 0.25 mg/mL sodium hyaluronatesolution (prepared by dissolving 100 mg of sodium hyaluronate (LifeCoreBiomedical) in 20.0 mL of SWFI. This was mixed by gently rotating and/orrocking at 2-8° C. for 2-4 hours, or until completely dissolved). Thereaction plate was transferred to the MULTIDROP 384 and the reaction wasinitiated by pressing the start key to dispense 30 μL sodium hyaluronateinto each well. The plate was then removed from the MULTIDROP 384 andshaken for 10 seconds before being transferred to a heat block with theplate cover replaced. The plate was incubated at 37° C. for 10 minutes.

The MULTIDROP 384 was prepared to stop the reaction by priming themachine with Serum Working Solution and changing the volume setting to240 μL. (25 mL of Serum Stock Solution [1 volume of Horse Serum (Sigma)was diluted with 9 volumes of 500 mM Acetate Buffer Solution and the pHwas adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM AcetateBuffer Solution). The plate was removed from the heat block and placedonto the MULTIDROP 384, and 240 μL of serum Working Solutions wasdispensed into the wells. The plate was removed and shaken on a platereader for 10 seconds. After a further 15 minutes, the turbidity of thesamples was measured at 640 nm and the hyaluronidase activity (in U/mL)of each sample was determined by fitting to the standard curve.

Specific activity (Units/mg) was calculated by dividing thehyaluronidase activity (U/ml) by the protein concentration (mg/mL).

B. Biotinylated Hyaluronan Assay

The biotinylated-hyaluronic acid assay measures the amount ofenzymatically active rHuPH20 or PEGPH20 in biological samples by thedigestion of a large molecular weight (˜1.2 megadaltons) biotinylatedhyaluronic acid (b-HA) substrate non-covalently bound to plasticmulti-well microtiter plates. The rHuPH20 or PEGPH20 in standards andsamples are allowed to incubate in a plate coated with b-HA at 37° C.After a series of washes, remaining uncleaved/bound b-HA is treated withStreptavidin Horseradish Peroxidase conjugate (SA-HRP). Reaction betweenimmobilized SA-HRP and the chromogenic substrate,3,3′,5,5′-tetramethylbenzidine (TMB), produces a blue colored solution.After stopping the reaction with acid, formation of the soluble yellowreaction product is determined by reading the absorbance at 450 nm usinga microtiter plate spectrophotometer. The decrease in absorbance at 450nm resulting from enzyme activity on the biotinylated hyaluronic acid(b-HA) substrate is a measure of the soluble rHuPH20 hyaluronidaseactivity. The method is performed using a calibration curve generatedwith dilutions of a soluble rHuPH20 or PEGPH20 reference standard, andsample activity measurements are made relative to this calibrationcurve.

Dilutions of the sample and calibrator were prepared in Assay Diluent.The Assay Diluent was prepared by adding 1% v/v pooled plasma (from theappropriate species) to 0.1% (w/v) BSA in HEPES, pH 7.4. This wasprepared daily and stored at 2-8° C. Depending upon the species type aswell as the anticipated hyaluronidase level, single or multipledilutions were prepared to ensure at least one sample dilution wouldfall within the range of the calibration curve. To guide the selectionof test sample dilution(s), information known about the dose ofhyaluronidase administered, the route of administration, approximateplasma volume of the species and the time point were used to estimatethe hyaluronidase activity levels. Each sample dilution was mixed as itwas prepared by brief pulse-vortexing and pipet tips were changed inbetween each dilution. In general, the dilutions began with an initial50 or 100-fold dilution followed by additional serial dilutions. Aseven-point calibration curve of rHuPH20 or PEGPH20 (depending upon thetreatment administered) was prepared ranging in concentration from 0.004to 3.0 U/mL for rHuPH20 and from 0.037 to 27 U/mL for PEGPH20.One-hundred microliters (100 μL) of each test sample dilution andcalibration curve point was applied to triplicate wells of a 96-wellmicrotiter plate (Immulon 4HBX, Thermo) that had been previously coatedwith 100 μL per well of b-HA at 0.1 mg/mL and blocked with 250 μL of1.0% (w/v) Bovine Serum Albumin in PBS. Plate(s) were covered with anadhesive plate seal and incubated at 37° C. for approximately 90minutes. At the end of the incubation period, the adhesive seal wasremoved from the plate, samples were aspirated and the plate washed five(5) times with 300 μL per well Wash Buffer (10 mM Phosphate Buffer, 2.7mM Potassium Chloride, 137 mM Sodium Chloride, pH 7.4, with 0.05% (v/v)Tween 20, PBST) using an automated plate washer (BioTek ELx405 SelectCW, Program ‘4HBX1’). One hundred microliters of Streptavidin-HRPConjugate Working Solution [Streptavidin-HRP conjugate (1:5,000 v/v) in20 mM Tris-HCl, 137 mM Sodium Chloride, 0.025% (v/v) Tween 20, 0.1%(w/v) Bovine Serum Albumin] was added per well. The plate was sealed andallowed to incubate at ambient temperature for approximately 60 minuteswithout shaking and protected from light. At the end of the incubationperiod, the adhesive seal was removed from the plate, samples wereaspirated and the plate washed five (5) times with 300 μL per well WashBuffer as described above. TMB solution (at ambient temperature) wasadded to each well and allowed to incubate protected from light forapproximately five (5) minutes at room temperature. TMB Stop Solution(KPL, Catalog #50-85-06) was then added as 100 μL per well. Theabsorbance of each well at 450 nm was determined using a microtiterplate spectrophotometer. The response of the Calibration Curve on eachplate was modeled using a 4-parameter logistic curve fit. Thehyaluronidase activity of each unknown was calculated by interpolationfrom the calibration curve, corrected for sample dilution factor, andreported in U/mL.

Example 5 Electrochemiluminescent Immunoassay for the Detection ofAnti-rHuPH20 Antibodies in Plasma

In this example, the presence of antibodies in plasma or serum raisedagainst the amino acid portion of PEGPH20 was measured using anelectrochemiluminescent (ECL) bridging assay (see Mire-Sluis et al.,Journal of Immunological Methods 289: 1-16, 2004).

A. Assay Overview

The method was typically performed in three Tiers. In Tier 1, an initialscreen was performed on the samples to be tested. Samples that yieldedECL values above a mathmatically determined cut point were tested inTier 2 while samples that did not yield ECL values above the cut pointwere reported as negative for anti-rHuPH20 antibodies. Tier 2 assaysconfirmed positive results from Tier 1 using an unlabled rHuPH20 as acompetitive inhibitor and Tier 3 assays were used to confirm at whatdilution of the sample the response remains above the cut point. Tier 1and Tier 2 assessments can be performed concurrently.

B. Preparation of Controls

A 1 mg/mL rabbit anti-rHuPH20 polyclonal antibody working stock wasgenerated by diluting 100 μl of 2.45 mg/mL rabbit anti-rHuPH20polyclonal antibody into 145 μl of StartingBlock T20 (TBS) BlockingBuffer (Thermo, catalog #37543). This stock was mixed thoroughly andsubsequently used to generate a second working stock (100 μg/mL) bydiluting 100 μl of the 1 mg/mL rabbit anti-rHuPH20 polyclonal antibodyinto 900 μl of StartingBlock T20 (TBS) Blocking Buffer (Thermo, catalog#37543). The 100 μg/mL working stock was then used to generate low, midand high positive controls as outlined in Table 5 below. The negativebase pool used was human K3-EDTA plasma (Bioreclamation, Catalog#HMPLEDTA). The diluent was StartingBlock Buffer. The negative controlis the negative base pool human plasma alone (unspiked). Baselinesamples from each dog were used as the control for the post-PEGPH20exposure samples.

TABLE 5 Positive and Negative Controls for the Anti-rHuPH20 AssayNominal Concentration Volume of Volume Total Concentration of WorkingWorking of Plasma Volume (ng/mL) Stock (ng/mL) Stock (μL) (mL) (mL)1,000 (High) 100,000 200 19.8 20 500 (Mid) 100,000 100 19.9 20 250 (Low)100,000 50 19.95 20 0 (Negative) 0 0 20 20 High, Mid, Low and Negativecontrols were pipetted into 100 μl aliquots and stored at −70° C. untiluse (for up to 1 year from date of preparation).C. Tier 1 Assay

Blood serum samples to be tested for the presence of anti-rHuPH20antibodies were collected and stored frozen at −70° C. until use. On theday of sample testing, high, mid, low and negative control samples(Example 5B) were removed from storage and thawed together with testsamples at ambient temperature, on ice, or at 2-8° C. until they werethawed. Test samples and control samples were mixed gently and eachdiluted 1:100 in StartingBlock T20 (TBS) Blocking Buffer (Thermo,catalog #37543) for a total of 200 μl per sample.

A separate 10 mL solution was prepared containing 250 ng/mL ofrHuPH20-Bt (Recombinant Human Hyaluronidase-Biotinylated; Millipore,special request) and 250 ng/mL of rHuPH20-Rt (Recombinant HumanHyaluronidase-Ruthenium; Millipore, special request) in StartingBlockT20 (TBS) Blocking Buffer. The solution was mixed gently and 200 μl wasadded to each of the 200 μl sample and control sample tubes. The tubeswere vortexed gently (Vortex Genie, Scientific Industries,Catalog#14-961-26), sealed and incubated overnight for 16-24 hours at2-8° C. with gentle rocking (Heidolph Rocking Platform Shaker, Model#Polymax 2040), protected from light.

During incubation, a Streptavidin Coated Standard MA2400 96 Plate (MesoScale Discovery, Catalog# L15SA) was blocked with 300 μl/wellStartingBlock (TBS) Blocking Buffer (Thermo, Catalog#37542) for 1-4hours at ambient temperature with gentle shaking. After blocking wascomplete, the StartingBlock (TBS) Blocking Buffer was removed byaspiration and the plate washed 3 times with 300 μl/well TBS-T washbuffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween, pH 7.4±0.1 atambient temperature) on a Bio-Tek Automated Plate Washer (Model#ELx405).

After aspirating the wash buffer, 100 μl/well of each sample and controlsample (each sample in duplicate) was added to a well of thestreptavidin coated plate and sealed with an opaque plate sealer (VWRInternational, Catalog#14230-062). Binding was allowed to proceed for30±3 minutes at ambient temperature with gentle shaking, protected fromlight.

During the 30 minute incubation, a 20 mL 1× solution of Read Buffer Twith Surfactant (4× Read Buffer; Meso Scale Discovery, Catalog# R92TC)was prepared with ultrapure water. When the sample incubation period wascomplete, the sample solution was aspirated from the plate (Bio-TekAutomated Plate Washer, Aspirate height 3.048 mm, Horizontal ApriratePosition 1.372 mm, Aspirate Flow Rate 3.0 mm/sec) and the plate washed 3times on a Bio-Tek Automated Plate Washer with 300 μl/well TBS-T washbuffer (Dispense Flow Rate 5, Dispense Height 15.24 mm). Read Buffer Twith Surfactant (1×) was then added to each well of the plate (150μl/well). Within 30 minutes of adding the Read Buffer T, the plate wasread on a Meso Scale Discovery Sector Imager 2400. Data(electrochemiluminescence units) was collected from the Sector Imager2400 using Meso Scale Discovery software (Gaithersburg, Md.).

(i) Data Validation

An assay was defined as samples analyzed together on a plate. A minimumof 3 negative controls were run (distributed as one near the front ofthe plate, one near the middle of the plate and one near the back of theplate, or as two near the front of the plate and two near the back ofthe plate) and 2 positive controls were run for each concentration(high, mid, low; one near the front of the plate and one near the backof the plate).

The system suitability specifications for each assay were establishedand documented on a “Test Method Summary Sheet”. An assay was acceptiblein Tier 1 assays if 1) The coefficient of variation (% CV) of theresponse (ECL units) of replicates for controls was ≦20.0% (only if theresponse value was >50 ECL units); 2) The ECL response values for ≧66.0%of the positive controls were within ±50% of the expected ECL responsevalue or the assigned acceptance range as specified on the Test MethodSummary Sheet; 3) The mean ECL response value for at least one positivecontrol was within ±50% of the expected ECL response value or theassigned acceptance range as specified on the Test Method Summary Sheetat each concentration level (i.e. low, mid, high controls); 4) The meanECL response value for at least one positive control was within ±50% ofthe expected ECL response value or the assigned acceptance range asspecified on the Test Method Summary Sheet in each set (front versusback of plate) of controls; 5) The mean ECL response value for thenegative controls was within the assigned acceptance range as specifiedon the Test Method Summary Sheet; and 6) The ECL response values for≧66.0% of the negative controls was within the assigned acceptance rangeas specified on the Test Method Summary Sheet.

An assay was acceptible in Tier 2 assays if in addition to the abovecriteria for Tier 1 assays, the assay also met the followingcriteria: 1) The percent inhibition for the positive controls was ≧50.0%for at least 50.0% of the positive controls at each concentration; 2)The percent inhibition for the positive controls was ≧50.0% for at least50.0% of the positive controls in each set (front versus back of theplate); and 3) The percent inhibition was ≧50.0% for ≧75.0% of allpositive controls.

A study sample was reanalyzed for any of the following reasons: 1) Anequipment malfuntion; 2) The test sample was lost during analyticalprocessing; 3) The % CV between replicate ECL values was >20.0% (Thiscriterion did not apply if the ECL responses of both of the samplereplicates were below the assay cut point, or if the ECL responses ofboth of the sample replicates were above the assay cut point. Inaddition, this criterion did not apply if the difference betweenreplicate ECL responses was 50 ECL units); 4) The mean ECL value of asample was the assay cut point in Tier 1 testing. The sample would beconsidered as a putative positive and submitted for Tier 2 testing,unless Tier 2 testing was performed concurrently; and 5) A documentedproblem associated with the study sample handling was discovered.

(ii) Data Analysis

A minimum of 3 negative controls were run in each tier. The individualvalues for the negative control were used to calculate the “floating cutpoint”. The “floating cut point” was calculated from each plate bymultiplying the geometric mean value of the 3 negative controls by aconstant normalization factor of 1.14 (derived from a parametricanalysis of the validation data). This floating cut point had a maximumvalue as determined by the “Test Method Assay Information Sheet”. If thecalculated cut point was greater than the maximum cut point listed inthe “Test Method Assay Information Sheet”, the maximum ECL units wasused as the cut point (maximum ECL value is evaluated for every lot ofnegative base pool).

Based on the cut point, each sample was evaluated. Samples with a meanECL value less than the cut point were reported as negative for rHuPH20antibodies. Samples with a mean ECL value greater than or equal to thecut point were classified as “Putative Positive” and subject to Tier 2testing (which can be performed concurrently). Samples with a mean ECLvalue greater than or equal to the cut point but had insuffient samplevolume to permit Tier 2 testing were reported as “Putative Positive” andnot tested further.

D. Tier 2 Assay

Each sample that was identified as being a putative positive in Tier 1,was submitted for Tier 2 testing. Tier 2 testing examined the ability ofan unlabeled excess of rHuPH20 to inhibit putative antibody binding tothe labeled rHuPH20.

Tier 2 testing was carried out essentially as for Tier 1 testing(Example 5C) except for the initial 1:100 sample dilution inStartingBlock T20 (TBS) Blocking Buffer. In Tier 2, each sample andcontrol sample were diluted 1:100 in StartingBlock T20 (TBS) BlockingBuffer (Thermo, catalog #37543) for a total of 200 μL and also diluted1:100 in StartingBlock T20 (TBS) Blocking Buffer containing 10 μg/mLrHuPH20 (created as a master mix of 8 mL StartingBlock T20 (TBS)Blocking Buffer with rHuPH20 at a concentration of 10 μg/mL) for a totalof 200 μL.

After reading the plate as in Example 5C, the percent of inhibition wascalculated according to the following equation: Percent Inhibition=(MeanECL value of the uninhibited sample) minus (mean ECL value of theinhibited sample) divided by (the mean ECL value of the uninhibitedsample) times 100. Any sample confirmed to have 50% inhibition orgreater was considered “Antibody Positive” and submitted for Tier 3testing. Samples with less than 50% inhibition were reported as“Antibody Negative”. Samples with 50% or greater inhibition but withinsufficient sample to continue to Tier 3 were reported as “AntibodyPositive” but not tested in Tier 3.

E. Tier 3 Assay

Each sample that was identified as being “Antibody Positive” in Tier 2,was submitted for titration in Tier 3. Tier 3 titration and testing wascarried out essentially as for Tier 1 testing (Example 5C) except thatsamples were diluted in StartingBlock T20 (TBS) initially 1:100 and thenserially diluted in Normal Plasma (as used for the negative control;Negative Base Pool).

After the dilution steps, each of the dilutions were further diluted1:100 in StartingBlock T20 (TBS) Blocking Buffer (as in Example 5C) togenerate the final assay dilution. The assay was then carried out as forTier 1.

After reading the plate, the titer (the highest sample dilution) thatyielded a mean ECL value above the assay cut point was determined.Titrations that had at least one dilution with a mean ECL value ≧ thecut point and one value < the cut point were reported as “AntibodyPositive” and the titer was reported as the final dilution where the ECLvalue was above the cut point. Titrations with all of the dilutions witha mean ECL value ≧ the cut point were reported as “Antibody Positive”and further dilutions were tested. Titrations where only the original1:100 dilution (as in Tier 1) had a mean ECL value ≧ the cut point werereported as “Antibody Positive −1:5”.

Example 6 Effect of PH20 on Musculoskeletal Effects

A. Cynomolgus Monkey

This Example describes the musculoskeletal observations that wereobserved in a 4-week repeat-dose toxicity study conducted in cynomolgusmonkeys following IV administration of PEGPH20. Four groups of monkeys(6 animals per gender, with the exception of the group that received 0.2mg/kg/dose of PEGPH20, which was 4 animals per gender) received IVtwice-weekly doses of vehicle, 0.2, 2.0 or 10.5 mg/kg/dose of PEGPH20,respectively, for 4 consecutive weeks. The twice-weekly IVadministration was well tolerated in monkeys, however changes in limbjoints were observed. Monkeys exhibited a dose-related decrease in rangeof motion at the knee and elbow, which showed partial to full recoveryfollowing cessation of dosing. Also, there was a moderate decrease insoft tissue mass (skeletal muscle) in a single high-dose animal observedby radiologic examination. This is consistent with the pharmacologiceffect of PEGPH20 to remove hyaluronan (HA) and its associatedextracellular water from tissues. There were no associatedhistopathologic changes (cartilage, tendons, ligaments) of the kneejoint or skeletal muscle, nor abnormal radiography findings of the kneejoint itself although movement range was limited. These observationsindicate that PEGPH20 administration can induce or result in transientmusculoskeletal effects.

B. Humans

This Example describes the results of a clinical study (Phase 1-101),assessing escalating dosage of PEGPH20 in patients to maintain elevatedenzyme levels in the plasma and limit return of HA substrate. The medianage of the patients was age 61 (range 56-86) with tumor types includinghistiocytoma, colorectal, pancreatic, bladder, carcinoid and ovarian. Inall cases, the patients entered the program under pain medicationrelated to their presenting condition but not for study drug-relatedpain.

1. Single Intravenous (IV) Dose of 0.05 mg/kg PEGPH20

Initially, two patients were administered a single intravenous (IV) doseof 0.05 mg/kg PEGPH20. This Example describes that both patientsexperienced stiffness and severe muscle and joint pain with an onset ofapproximately 6-10 hours after dosing. The pain lasted more than 9 dayswith patients experiencing stiffness, muscle and joint pain and weaknessthat interfered with activities of daily living.

The first patient received an intravenous (IV) dose of 4.6 mg (0.05mg/kg) of PEGPH20 (patient mass of 92 kg). Approximately 10 hourspost-dose, the patient reported bilateral hip pain, with difficultywalking and a moderately severe sore throat. The patient wasdiscontinued from the study after the single dose. On Day 2, the patientreported being unable to get out of bed due to muscle and joint pain andstiffness in the upper and lower extremities. The upper limbs could bemoved, with the elbow and wrist joints able to flex and extend, howeverthe knees were stiff and were not able to fully extend. The severity ofthe muscle/joint pain was Grade 3. Over the course of 3 weeks, thepatient showed slow but steady improvement of the musculoskeletaleffects.

The second patient received an IV dose of 4.9 mg (0.05 mg/kg) of IVPEGPH20 (patient mass of 98 kg). Between 4 and 8 hours post-dose, thepatient reported musculoskeletal pain. The patient was discontinued fromthe study after the single dose. On Day 2, the patient reported profoundpain, initially in the knees, but also in all muscles and bones and asore throat. The severity of the muscle/joint pain was Grade 3. By Day5, the patient reported feeling better and by Day 9 the patient reportedthe musculoskeletal pain to have substantively improved.

The patient plasma samples also were analyzed using a human inflammationmulti-analyte profile (MAP) of 46 different cytokines, chemokines,acute-phase reactants, and other plasma inflammatory biomarkers. Nonotable findings were observed for the first two patient samples, rulingout general inflammation or muscle damage.

2. Twice Weekly Intravenous (IV) Dose at 0.5 μg/kg PEGPH20

Given the severity of the symptoms of the first two patients, a thirdpatient was dosed at 0.0005 mg/kg (0.5 μg/kg) twice weekly. On Day 1,this patient received an IV dose of 0.06155 mg PEGPH20. On Day 3, thepatient experienced a one to two minute, spontaneously resolving crampin his right calf. The patient received the second IV dose of 0.06155 mgPEGPH20 on Day 4. On Day 5, the patient reported repeated muscle crampsin his feet, calves, and thighs, which lasted approximately three tofour minutes and occurred approximately six to eight times throughoutthe day. The cramps were accompanied by muscle soreness and tendernessover the entire body but with no joint involvement. The patient wasambulatory but stiff. The severity of the muscle/joint pain was Grade 3.Due to these musculoskeletal effects, the patient met dose-limitingtoxicity and was given no further doses of PEGPH20. The muscletenderness resolved by Day 21 and the muscle cramps by Day 43.

Hyaluronidase enzyme levels and HA catabolite levels also were measuredas indicated above in Example 6B.1. A summary of mean enzyme levels andHA catabolites following dosing with 0.5 μg/kg PEGPH20 also is set forthin Example 11.

3. Single Intravenous (IV) Dose of 0.5 μg/kg PEGPH20 Every 21 days

Three additional patients were also treated with an IV dose of 0.5 μg/kgPEGPH20 every 21 days. Two of the patients received a second dose of 0.5μg/kg PEGPH20, 21 days after the first dose. Only intermittent Grade 1or transient Grade 2 musculoskeletal side effects observed in allpatients. No dose-limiting toxicities (DLTs) occurred in this dosecohort. Nevertheless, all patients were discontinued due to diseaseprogression.

Hyaluronidase enzyme levels and HA catabolite levels also were measuredas indicated above in Example 6B.1. A summary of mean enzyme levels andHA catabolites following dosing with 0.5 μg/kg PEGPH20 also is set forthin Example 11.

4. Single Intravenous (IV) Dose of 0.75 μg/kg PEGPH20 Every 21 Days

Given the attenuation of musculoskeletal effects at the IV dose of 0.5μg/kg PEGPH20 every 21 days, the dose of PEGPH20 was increased. Fourpatients were subsequently given an IV dose of 0.75 μg/kg PEGPH20 every21 days, and the dosage regimine in this cohort included up to threedoses were given at this dose level. Generally, only intermittent Grade1 or transient Grade 2 musculoskeletal side effects observed. Allpatients were discontinued due to disease progression. A summary of theresults of each of the patients is as follows:

The first of the patients had moderately differentiated pancreaticadenocarcinoma and experienced Grade 1 musculoskeletal pain two daysafter the first cycle 2 dose. The event severity increased to Grade 3after 10 days. This was not considered a dose-limiting toxicity, sinceit occurred after the first cycle. The event lasted a total of 14 days(10 days at Grade 1 and four days at Grade 3) and resolved. This patientwas withdrawn after two cycles due to disease progression.

The second patient in the cohort had small bowel mesenteric carcinoidtumor and experienced Grade 1 hand cramping seven days after the Cycle 1dose. The patient was discontinued due to disease progression.

The third patient had ovarian adenocarcinoma and experienced a Grade 2adverse event of bone pain (sacrum, sternum and knees) and muscle acheson the same day as cycle 1 dose. The muscle aches resolved after 10days, and the bone pain decreased in severity to Grade 1 after anadditional 10 days and resolved after a total of 49 days. That patientdiscontinued due to disease progression after three cycles. In thispatient, cancer antigen-125 (CA-125) was measured, which is a proteinthat is found at levels in ovarian cancer cells that are elevatedcompared to normal cells. Notably, CA-125 decreased from 103 U/mL (Cycle1 Day 1) to 64 U/mL (Cycle 1 Day 15). On Cycle 3 Day 1, which was thelast value recorded, CA125 was 116.4 U/mL.

The fourth patient had colon adenocarcinoma and received a total of twocycles. Grade 1 muscle spasms occurred in the upper back two days afterthe first dose and lasted one day. Grade 1 bilateral hand, knee andshoulder stiffness occurred nine days after the second dose.

Hyaluronidase enzyme levels and HA catabolite levels also were measuredas indicated above in Example 6B.1. A summary of mean enzyme levels andHA catabolites following dosing with 0.75 μg/kg PEGPH20 also is setforth in Example 11.

5. Single Intravenous (IV) Dose of 1.0 μg/kg PEGPH20 Every 21 Days

The dose of PEGPH20 was increased 33%, and three patients were given anIV dose of 1.0 μg/kg PEGPH20 every 21 days. Two of the patients hadprostate adenocarcinoma and one patient had Non Small Cell Lung Cancer[NSCLC]. There were no dose-limiting toxicities (DLTs) reported. Onlyone of the patients reported musculoskeletal symptoms, which werecategorized as Grade 1 intermittent muscle spasms (ribs, feet, calves),intermittent joint pain and rib pain. The symptoms all started one tothree days after the Cycle 1 dose. The rib pain resolved after ninedays, the muscle spasm resolved after 43 to 44 days and the hip painresolved after 45 days.

6. Single Intravenous (IV) Dose of 1.5 μg/kg PEGPH20 Every 21 Days

One patient, with prostate adenocarcinoma, received an IV dose of 1.5μg/kg PEGPH20 every 21 days. There were no dose-limiting toxicities(DLTs) reported.

7. Summary

Of the 14 patients treated, 3 were discontinued due to adverse events:one receiving 50.0 μg/kg dose twice weekly due to Grade 4musculoskeletal pain, one receiving 50.0 μg/kg dose twice weekly due toGrade 3 musculoskeletal pain, and one receiving 0.5 μg/kg in a 21 daycycle due to Grade 3 musculoskeletal pain. One patient, receiving 0.5μg/kg in a 21 day cycle, completed two cycles of therapy and then wasdiscontinued due to disease progression. All other patients werediscontinued due to disease progression.

Example 7 Effect of PEGPH20 On Musculoskeletal Effects in Beagle Dogs

A. Tolerability and Pharmacokinetics Dose Range Study in Beagle Dogs

In light of the musculoskeletal observations in monkeys and humans inresponse to IV PEGPH20, a non-clinical model for these musculoskeletaleffects was sought. Due to their docile temperment and ease of handlingfor procedures and assessments, the beagle dog (BioTox Sciences ContractResearch Organization, San Diego, Calif. using BioTox Sciences colonydogs that originated from Marshall Farms USA, Inc., North Rose, N.Y.)was assessed for its ability to model musculoskeletal symptoms similarto those observed in humans, in response to PEGPH20 administration. Atolerability and pharmacokinetics study of single intravenous doses ofPEGPH20 in beagle dogs was evaluated. A dose range-finding study wasperformed to evaluate intravenous (IV) doses of PEGPH20 from 3040 to47500 Units/kg (0.08 to 1.25 mg/kg).

On Day 1, one male beagle dog was dosed with a bolus injection of 5 mLvia percutaneous needle puncture of the cephalic vein with PEGPH20 atthe initial dose of 3040 Units/kg (0.08 mg/kg). The dog appeared visablynormal until Day 2 when it was observed to exhibit reduced mobility(reduced ability to walk or stand) and tighness of muscles of the neck,back and extremities upon palpation. At 48 hours post-dose, the mobilityof the animal had improved and by Day 3, the animal exhibitednear-complete recovery. Based on these observations of severemusculoskeletal responses to PEGPH20, the dose escalation study washalted.

B. Musculoskeletal Response to IV PEGPH20 in Beagle Dogs

To confirm and further evaluate the musculoskeletal response to PEGPH20,three male beagle dogs were dosed with 80 mg/kg (3040 U/kg) of PEGPH20,two intravenously (IV) and one subcutaneously (SC), and visuallyobserved for musculoskeletal responses (Table 6). PEGPH20 wasadministered as a 5 mL/animal IV or SC bolus injection. IVadministration was via percutaneous needle puncture of the cephalicvein, whereas SC administration was via percutaneous needle punctureinto the dorsal back region, near the shoulders. By 23 hours post-dose(Day 2), all three dogs exhibited reduced mobility (reduced ability towalk or stand), overall decreased activity, and muscle stiffness in theneck, back and extremities upon palpation. By Day 4, each of themusculoskeletal responses had resolved.

TABLE 6 Schedule of Single Dose IV Administration of PEGPH20 Dose[Units/kg (mg/kg)] Study DOG 989 DOG 153 DOG 288 Day (IV dose) (SC dose)(IV dose) 1 3040 U/kg 3040 U/kg 3040 U/kg (0.08 mg/kg) (0.08 mg/kg)(0.08 mg/kg) 2 0 0 0 3 0 0 0

This study confirmed the musculoskeletal findings of the study describedin Example 7A and established the Beagle dog as a non-clinical model forPEGPH20-mediated musculoskeletal effects similar to those observed inhumans.

C. Repeat PEGPH20 Administration in Beagle Dogs

To investigate the feasibility of repeated administration of PEGPH20 tocondition dogs to tolerate subsequent doses of PEGPH20, the three dogsdosed in Example 7B were redosed IV, through the cephalic vein orsubcutaneously (SC) via percutaneous needle puncture into the dorsalback region near the shoulders with daily escalating PEGPH20 doses from1520 to 60800 Units/kg (0.04 to 1.6 mg/kg) according to the scheduleshown in Table 7.

TABLE 7 Schedule of Repeat Administration of PEGPH20 with EscalatingDoses of PEGPH20 Dose [Units/kg (mg/kg)] Study DOG 989 DOG 153 DOG 288Day (IV dose) (SC dose) (IV dose) 4  1520 (0.04)  1520 (0.04)  1520(0.04) 5  2280 (0.06)  2280 (0.06)  2280 (0.06) 6  3040 (0.08)  3040(0.08)  3040 (0.08) 7  3800 (0.1)  3800 (0.1)  3800 (0.1) 8  7600 (0.2) 7600 (0.2)  7600 (0.2) 9 15200 (0.4) 15200 (0.4) 15200 (0.4) 10 22800(0.6) 22800 (0.6) 22800 (0.6) 11 30400 (0.8) 30400 (0.8) 30400 (0.8) 1260800 (1.6) 60800 (1.6) 60800 (1.6)

On Days 4 through 6, all three dogs exhibited musculoskeletal responsesto PEGPH20 including reduced mobility (reduced ability to walk orstand), overall decreased activity, and muscle stiffness in the neck,back and extremities upon palpation. While the onset of themusculoskeletal responses on Days 4 through 6 was more rapid (a fewhours) than the musculoskeletal responses seen on Day 1 (23 hours), theseverity of the responses decreased from Day 4 to Day 6. In addition,recovery from the responses was also more rapid on Days 4 through Day 6(up to 12 hours) than observed on Day 1 (2 days). No musculoskeletalresponses were observed in all three dogs on Day 7 through Day 9. On Day12, one dog dosed IV (dog 288), exhibited reduced mobility 2 hours afterthe 60800 Units/kg (1.6 mg/kg) dose with complete recovery within 10hours post-dose. This same dog also exhibited retching post-dose on Day10 and retching and vomiting on Day 11 and Day 12.

D. Effect of Immunogenicity of PEGPH20 in Beagle Dogs

Multiple repeated dosing following a break in dosing was examined in thethree male beagle dogs from Example 7C above. On Day 17, 26 or 33, onedog received 3040 Units/kg (0.08 mg/kg) PEGPH20 intravenously with a 5mL bolus injection via percutaneous needle puncture of the cephalic veinaccording to the schedule in Table 8.

TABLE 8 Schedule of PEGPH20 Dosing Dose [Units/kg (mg/kg)] Study Day DOG989 DOG 153 DOG 288 17 3040 (0.08) 26 3040 (0.08) 33 3040 (0.08)

Immediately post-dose, each of the three dogs was observed to experiencea systemic anaphylactoid-like reaction. Reactions included urination,defecation, decreased physical activity, difficulty ambulating, retchingand/or vomiting, and increased breathing frequency and depth. Markedrecovery occurred within 10 to 25 minutes with complete recovery within24 hours post-dose. No further changes in animal mobility, activity ormuscle tone were observed at any subsequent post-dose time points(animals were observed twice a day until the next dosing event).

Due to the possibilty that the reactions noted above were caused by animmunologic repsonse to the administered PEGPH20 (a human protein), serafrom the pre-dose bleeds on Day 17, 26 or 33 and from bleeds from allthree dogs on Day 51, were tested for reactivity to recombinant humanhyaluronidase (rHuPH20) to establish whether the dogs had mounted ahumoral immune response to the PEGPH20.

On Days 17, 26 or 33, blood was drawn before dosing (but after the 12days of excalating PEGPH20 exposure on Day 1-Day 12), from the dog to betreated on that day. Blood was also drawn from all three dogs on Day 51.Serum isolated from blood was tested for the presence of anti-rHuPH20antibodies as described in Example 5 above. The results of the assay areshown in Table 9. These results confirm that all three dogs mounted astrong antibody response to the protein component, rHuPH20, of PEGPH20,indicating that PEGPH20 is immunogenic in beagle dogs. This finding isnot unexpected since the protein component of PEGPH20 is a humanprotein.

TABLE 9 Anti-rHuPH20 Serum Antibodies Following Repeat Dosing in BeagleDogs Post-PEGPH20 Post-PEGPH20 Animal Baseline Exposure Exposure ID No.(predose Day 1) (Variable Days) (Day 51) 989 Not Detected Day 17:1:125,000 1:25,000 153 Not Detected Day 26: 1:125,000 1:625,000 288 NotDetected Day 33: 1:78,125,000 1:1,953,125,000

To examine if the antibody response in Beagle dogs lead to a potentialclearing or neutralization of the administered PEGPH20, the ability ofthe re-dose of PEGPH20 to deplete hyaluronan from skeletal muscle wasexamined.

The same three dogs (989, 153 and 288) were treated with an IV dose of3040 Units/kg (0.08 mg/kg) PEGPH20 on Day 59. Immediately post-dose, oneanimal (dog #288) exhibited retching with a complete recovery by 5minutes. No musculoskeletal effects were observed at any subsequent timepoint.

Twenty-four hours post-dose (Day 60), skeletal muscle biopsies weretaken from each of the three treated dogs and also from an untreatedcontrol beagle dog. The medial semitendinosis/medial membranosusmuscle(s) were targeted for biopsies. The area of the biopsy was shavedusing electric clippers and scrubbed using either Chlorhexidine orPovidone-Iodine solutions. Bupivacaine or Lidocaine (2-3 mg/kg) wasinjected subcutaneously (SC) in the area of the biopsy for localanalgesia. The muscle biopsy was collected using a 2-mm-diameter biopsyneedle. A gauze sponge was applied with slight pressure to stop anybleeding. Metacam (meloxicam) 0.2 mg/kg was injected SC to control pain.Biopsy samples were placed in Eppendorf tubes or equivalent withapproximately 200 μL 10% neutral-buffered formalin (NBF).

Biopsied tissue samples were stained for tissue HA using a highlyspecific histochemical staining method. Using this method, tissuebiopsies fixed in 10% NBF for 48 hours, were embedded in paraffin,sectioned and probed for HA using a biotinylated HA-binding protein(HABP; Seikagaku, Japan), then probed with FITC-labeled streptavidin(Vector Labs, Canada) for detection. The cell nuclei were counterstainedwith DAPI (4′,6-diamidino-2-phenylindole) reagent. Micrographs were thencaptured using a Zeiss Microscope (Thornwood, N.Y.) coupled with theSpot imaging program (Diagnostic Instruments, Inc).

Hyaluronan expression in the sections was evaluated visually by thelevel of green fluorescent intensity present. Hyaluronan staining of thepericellular matrix of skeletal muscle samples taken from all threePEGPH20 treated animals was virtually the same high intensity stainingas the hyaluronan staining in tissue samples from untreated controlanimals revealing that PEGPH20 treatment in these animals did not removeextracellular hyaluronan in skeletal muscle samples. This indicates thatthe serum anti-rHuPH20 antibodies generated in the beagle dogs inresponse to repeated PEGPH20 administration, likely possessed PH20neutralizing activity.

Example 8 Effect of Dexamethasone on PEGPH20 in Beagle Dogs

The beagle dog was identified as a species that exhibits similarmusculoskeletal observations, in terms of their presentation and timingfor onset and resolution, to those reported in humans, in response toPEGPH20 administration. Therefore, the beagle dog was further exploredas a model of the human musculoskeletal responses to PEGPH20 and as amodel to examine the treatment of symptoms.

A. Premedication Regimen for Dexamethasone

Studies were carried out to optimize the premedication regimen fordexamethasone.

In one study, four naïve male beagle dogs were orally dosed with 4mg/dose dexamethasone twice a day on the day of PEGPH20 administration.The first dexamethasone dose was given orally (PO) immediately beforePEGPH20 intravenous (IV) administration. The second dexamethasone dosewas given orally ˜8 hours later. On day 1, one dog was dosed with 3040U/kg (0.08 mg/kg) PEGPH20 after Dexamethasone. Twenty four hours later(day 2), a second dog was dosed with 5700 U/kg (0.15 mg/kg) PEGPH20after Dexamethasone. Twenty four hours later (day 3), a third dog wasdosed with 11400 U/kg (0.3 mg/kg) PEGPH20 after Dexamethasone. Twentyfour hours later (day 4), a fourth dog was dosed with 38000 U/kg (1.0mg/kg) PEGPH20 after Dexamethasone. Blood was collected pre-PEGPH20administration, and 0.5 hr, 4 h and 24 h post-PEGPH20 administration.Animals were observed for musculoskeletal effects pre-dose and 0.5, 4, 8and 24 hours post-dose. None of the 4 dogs developed musculoskeletalresponses to PEGPH20 at any dose level at any of the time points tested,indicating that dexamethasone premedication blocked the development ofmusculoskeletal effects of PEGPH20.

B. Effect of Dexamethasone On PEGPH20-Induced MusculoskeletalObservations

The ability of dexamethasone to ameliorate the musculoskeletal effectsof PEGPH20 was confirmed in a further pharmacological study. The fourtreatment groups (each with 3 beagle dogs) and the days of treatment areshown in Table 10 below. PEGPH20 was administered IV with a 5 mL bolusinjection via percutaneous needle puncture of the cephalic vein on studyDays 1, 2 and/or 5. Dexamethasone was administered orally immediatelybefore the PEGPH20 injection and approximately 8 hours post injection.All animals were re-dosed 3 days after the first injection except thosein the PEGPH20 alone treatment group, which were not re-dosed.

TABLE 10 Study Design of Dexamethasone Premedication Regimen Treatment #of Medication Received on Indicated Study Day Group dogs 1 2 4 5 VehicleControl 3 Vehicle Control Vehicle Control (API Buffer) (API Buffer)PEGPH20 alone 3 PEGPH20 (0.3 mg/kg) PEGPH20 (low) + 3 PEGPH20 (0.3mg/kg) + PEGPH20 (0.3 mg/kg) + Dexamethasone Dexamethasone (4 mgDexamethasone (4 mg before PEGPH20 and 4 mg, before PEGPH20 and 4 mg, 8hr after PEGPH20) 8 hr after PEGPH20) PEGPH20 (high) + 3 PEGPH20 (1.0mg/kg) + PEGPH20 (1.0 mg/kg) + Dexamethasone Dexamethasone (4 mgDexamethasone (4 mg before PEGPH20 and 4 mg, before PEGPH20 and 4 mg, 8hr after PEGPH20) 8 hr after PEGPH20)

Dogs were observed for musculoskeletal response to PEGPH20 immediatelyafter treatment with the first dose of PEGPH20 and also at 0.5, 2, 4, 7,10, 12, 16, 20, 24, 48, and 72 hours post-dose. After the second dose,animals were observed at 0.5, 2, 4, 7, 10, 12, 16, 20, 24, 48, 72, 96and 120 hours post second dose. Animals treated with only one dose ofPEGPH20 were evaluated at 96 hours, 120 hours, 6, 7, 8, and 9 dayspost-dose.

Beagle dogs administered PEGPH20 (11400 Units/kg; 0.3 mg/kg) aloneshowed moderate to severe musculoskeletal observations characterized byreduced mobility (reduced-ability to walk or stand), overall decreasedactivity and muscle stiffness of the neck, back and extremities uponpalpation. In general, the onset of these musculoskeletal observationswas first observed approximately 10 hours post-PEGPH20 dose, graduallyincreased in severity between 12-20 hours post-PEGPH20 dose and weresubsequently completely resolved by approximately 72 hours post-PEGPH20dose. Dexamethasone pre-treatment ameliorated these musculoskeletalsymptoms in dogs dosed with PEGPH20 at either 0.3 mg/kg or 1.0 mg/kg atall time points examined.

C. Effect of Dexamethasone on PEGPH20 Hyaluronan Removal

Given that dexamethasone pretreatment ameliorated PEGPH20-inducedmusculoskeletal responses, the effects of dexamethasone on PEGPH20removal of hyaluronan were examined in skeletal muscle and skin tissues.

Skeletal muscle and skin tissues were biopsied from each of the threedogs in each treatment group outlined in Table 11, 24 hours before and24 hours after the first IV dosing of PEGPH20. The medialsemitendinosis/medial membranosus muscle(s) were targeted for biopsies.The area of the biopsy was shaved using electric clippers and scrubbedusing either Chlorhexidine or Povidone-Iodine solutions. Bupivacaine orLidocaine (2-3 mg/kg) was injected subcutaneously (SC) in the area ofthe biopsy for local analgesia. The muscle biopsy was collected using a2-mm-diameter biopsy needle. A gauze sponge was applied with slightpressure to stop any bleeding. Metacam (meloxicam) 0.2 mg/kg wasinjected SC to control pain. Biopsy samples were placed in Eppendorftubes or equivalent with approximately 200 μL 10% neutral-bufferedformalin (NBF).

Biopsied tissues samples were stained for tissue HA. Briefly, tissuebiopsies fixed in 10% NBF for 48 hours, were embedded in paraffin,sectioned and probed for HA using a biotinylated HA-binding protein(HABP; Seikagaku, Japan), then probed with FITC-labeled streptavidin(Vector Labs, Canada) for detection. The cell nuclei were counterstainedwith DAPI (4′,6-diamidino-2-phenylindole) reagent. Micrographs were thencaptured using a Zeiss Microscope (Thornwood, N.Y.) coupled with theSpot imaging program (Diagnostic Instruments, Inc).

Hyaluronan expression in the sections was evaluated visually by thelevel of green fluorescent intensity present. The skeletal muscle andskin tissues from animals treated with Vehicle Control (API buffer)showed a similar pattern of brightly stained pericellular hyaluronan inboth pre-treatment and post-treatment samples. While skeletal muscle andskin tissues from animals treated with PEGPH20 (0.3 mg/kg) alone,PEGPH20 (0.3 mg/kg)+dexamethasone (4 mg/dose BID; twice a day) andPEGPH20 (1.0 mg/kg)+dexamethasone (4 mg/dose BID; twice a day) eachshowed brighly stained pericellular hyaluronan in pre-treatment samples,the post-treatment samples from each of these treatment groups revealedvirtually no hyaluronan staining. These findings indicated thatdexamethasone did not inhibit the hyaluronan degradating activity ofPEGPH20.

D. Effect of Dexamethasone On PEGPH20 Pharmacokinetics

To examine the effects of dexamethasone on PEGPH20 pharmacokinetics,blood samples were collected from each dog from Example 8B, in Table 10above. Blood was drawn after dosing with PEGPH20 or Vehicle Controlaccording to Table 11 below.

TABLE 11 Time points of Blood Draws for Pharmacokinetic MeasurementsTime point of Blood Draw per Treatment Group Vehicle Control PEGPH20(0.3 mg/kg) + PEGPH20 (1.0 mg/kg) + (API Buffer) PEGPH20 (0.3 mg/kg)Dexamethasone (4 mg; BID) Dexamethasone (4 mg; BID) Post First 5 min, 10min, 30 min, 5 min, 10 min, 30 min, 2 hr, 5 min, 10 min, 30 min, 5 min,10 min, 30 min, Dose 2 hr, 4 hr, 7 hr, 10 hr, 4 hr, 7 hr, 10 hr, 24 hr,48 2 hr, 4 hr, 7 hr, 10 hr, 2 hr, 4 hr, 7 hr, 10 hr, 24 hr, 48 hr, 72 hrhr, 72 hr, 96 hr, 120 hr, 6 24 hr, 48 hr, 72 hr 24 hr, 48 hr, 72 hrdays, 7 days, 8 days, 9 days Post Second 10 min, 30 min, 2 hr, 4 hr, N/A10 min, 30 min, 2 hr, 4 hr, 10 min, 30 min, 2 hr, 4 hr, dose 7 hr, 10hr, 24 hr, 48 hr, 7 hr, 10 hr, 24 hr, 48 hr, 7 hr, 10 hr, 24 hr, 48 hr,72 hr, 96 hr, 120 hr 72 hr, 96 hr, 120 hr 72 hr, 96 hr, 120 hr

Plasma Hyaluronidase activity was measured at each time point using thebiotinylated-hyaluronic acid assay described in Example 4B. Plasma wasprepared from collected blood samples and stored frozen at −70° C. untilanalysis. The lower limit of quantitation for the assay was 2.90 U/mL.Plasma concentration versus time data was analyzed by non-compartmentaland compartmental methods using WinNonlin Pro version 5.1 (PharsightCorp., Mountain View, Calif.). Derived PK parameters included AUC, Cmax,and Tmax. Systemic exposure defined by AUC and Cmax was similar betweenPEGPH20 alone and PEGPH20+dexamethsone when administered intravenouslyto dogs and the general PK profiles from the three PEGPH20 treatmentgroups were similar indicating that dexamethasone does not affect thepharmacokinetics of PEGPH20.

HA catabolites also were measured as described in Example 6. The resultsshow the appearance of HA catabolites following administration byPEGPH20, which was not affected by the presence of dexamethasone.

Example 9 Effect of Dexamethasone on the Antitumor or HyaluronanDegrading Activities of PEGPH20 in Human Prostate or Human PancreaticTumor Xenograft Models

To examine if dexamethasone interfered with the antitumor or hyaluronandegrading activities of PEGPH20, human prostate cancer or humanpancreatic cancer xenograft models were evaluated.

A. PC3 Prostate Cancer Xenograft Models

1. Antitumor Activity in PC3 Prostate Cancer Xenograft Models

Tumor cells from the PC3 prostate carcinoma cell line were grown toapproximately 80% confluency then trypsinized, collected, washed once inHBSS (Hank's balance salt solution, Mediatech Inc.), then re-suspendedin 50% Matrigel® in HBSS at 2×10⁷ cells/mL on ice before inoculationinto animals. Athymic male nude mice (Nu/Nu (Ncr); Taconic Farms;average weight ˜20 g) were inoculated intramuscularly (IM) with 0.05 mLof this cell suspension, peritibially, in the left hind leg (adjacent tothe tibia periosteum). The implants in the animals were assessed twice aweek and allowed to grow to a mean tumor volume of approximately 500 mm³(Day −2; Table 12). Actual tumor volumes were determined usingVisualSonics Vevo 770 high-resolution ultrasound, using twoperpendicular axial dimensions.

Animals were sorted randomly and grouped into 6 groups of 8 mice pergroup (6+2 satellite mice). Treatment groups were vehicle (API buffer),PEGPH20 (157,500 Units/kg, 4.5 mg/kg), low dose dexamethasone (1.25mg/kg), high dose dexamethasone (5 mg/kg), PEGPH20 (4.5 mg/kg)+low dosedexamethasone (1.25 mg/kg) and PEGPH20 (4.5 mg/kg)+high dosedexamethasone (5 mg/kg). The dose and frequency of the PEGPH20 (orvehicle) intravenous injections was 157,500 Units/kg or approximately4.5 mg/kg, every 3^(rd) day starting on Day 0 (Days 0, 3, 6, 9, 12) witha total of 5 injections (Q3Dx5) with a dose volume of 0.1 mL. Thisdosing and frequency regimen was previously shown to inhibit xenografttumor growth by 34-70%. The doses and frequency of the dexamethasoneintraperitonal injections were 1.25 mg/kg at the low dose and 5 mg/kg atthe high dose, once a day until Day 13 (QDx13) with a dose volume of 0.1mL.

The tumor volumes of all mice were measured pre-treatment on Day −2(average tumor volume of ˜500 mm³) and on Day 1, 6, 9, 13, 16 and 20 bycapturing images using the VisualSonic ultrasound system and using anultrasound imaging software program to calculate tumor volume (Table12). The percent tumor growth inhibition (% TGI) was calculated by thefollowing equation:[1−(T_(n)−T₀)/(C_(n)−C₀)]×100%In the above equation, T_(n) is the average tumor volume in thetreatment group at respective day “n” at the indicated timepoint aftertreatment; T₀ is the average tumor volume in the treatment group at Day0 before treatment; C_(n) is the average tumor volume in the controlgroup at respective day “n” at the indicated timepoint after treatmentwith vehicle; and C₀ is the average tumor volume in the control group atDay 0 before treatment.

The data in Table 1.2 show that dexamethasone alone had no effect ontumor growth inhibition, with either the low or high dose. The resultsalso show that the effects on tumor growth was similar in the groupstreated with PEGPH20 alone versus PEGPH20 and dexamethasone. Thus, thedata from the PC3 prostate cancer xenograft models revealed thatdexamethasone did not interfere with the effects of PEGPH20 on tumorgrowth inhibition

TABLE 12 Tumor Volumes and Statistical Analysis of Antitumor Activity ofPEGPH20, Dexamethasone as Single-agents and in Combination in PC3Prostate Cancer Model Day Group Day −2 Day 1 Day 6 Day 9 Day 13 Day 16Day 20 API-buffer 487.3 653.1 861.5 994.5 1252.7 1528.8 1870.5 SE 51.5770.03 56.14 71.78 91.28 95.31 144.22 PEGPH20 4.5 mg/kg 491.10 394.38598.39 674.83 827.53 1046.73 1310.85 SE 49.26 47.61 69.29 76.03 102.22119.17 177.85 p-value 0.004 0.005 0.004 0.004 0.01 0.02 % TGI TR 71 6456 47 41 Dexamethasone 1.25 mg/kg 486.88 636.98 727.80 964.35 1202.351401.91 1792.92 SE 48.16 66.17 96.52 85.93 119.73 130.68 166.24Dexamethasone 5 mg/kg 516.25 633.84 854.90 995.47 1211.50 1455.271840.11 SE 51.14 56.61 69.60 91.98 125.39 207.27 257.18 Dexamethasone1.25 mg/kg + 489.69 491.36 673.05 766.85 914.62 1067.69 1498.98 PEGPH204.5 mg/kg SE 45.71 62.92 78.57 83.52 98.62 148.24 223.68 p-value 0.050.04 0.029 0.012 0.01 % TGI 99 51 45 44 45 Dexamethasone 5 mg/kg +493.92 471.88 661.93 693.24 839.57 995.19 1343.58 PEGPH20 4.5 mg/kg SE44.40 54.47 58.26 60.04 71.02 63.90 76.21 p-value 0.03 0.01 0.003 0.0020.0005 0.005 % TGI TR 55 61 55 52 39 SE = Standard Error; % TGI = %Tumor Growth Inhibition; TR = Tumor Regression; p-value = One-tailedPaired Equal Variance Student's T-test

2. Effect of Dexamethasone on PEGPH20 Plasma Enzyme

Plasma samples were also collected from individual PC3 tumor-bearingsatellite mice of control and treatment group mice 24 hours after thefinal PEGPH20 and dexamethasone dose at Day 13. Plasma PEGPH20Hyaluronidase was assessed using the biotinylated-hyaluronic acid assaydescribed in Example 4B. Animals treated with PEGPH20, PEGPH20+low dosedexamethasone and PEGPH20+high dose dexamethasone all had similar levelsof plasma hyaluronidase activity indicating that dexamethasone did notinterfere with the level of PEGPH20 activity in the plasma of mice.

Plasma samples were additionally used to measure soluble plasmahyaluronan in the control group and each of the treatment groups (R&DSystems Catalog DY3614; ELISA assay). In the vehicle control group, theplasma hyaluronan levels ranged from 800,000 to 900,000 ng/mL. The lowdose dexamethasone group had a high range of variability in plasmahyaluronan concentrations (one high concentration and one lowconcentration but both measurable) but the high dose dexamethasone grouphad plasma hyaluronan levels ranging between 23,000 and 42,000 ng/mL.Mice treated with PEGPH20 alone or with PEGPH20+dexamethasone (eitherhigh or low dose) had no detectable (below limit of quantitation)hyaluronan demonstrating that dexamethasone did not alter the PEGPH20plasma activity in mice.

Thus, the results show that dexamethasone had no effect on PEGPH20activity in the plasma.

3. Effect of Dexamethasone on PEGPH20 Hyaluronan Degradation Activity inPC3 Prostate Cancer Xenograft Models

Tumor tissues from the two satellite mice from all 6 treatment groups inExample 9A.1 above, were harvested by tissue biopsy on Day 14 andstained for the presence of hyaluronan. Tumor biopsies were fixed in 10%NBF for 48 hours, embedded in paraffin, and sectioned. Samples wereprobed for hyaluronan using a biotinylated HA-binding protein (HABP),then probed with FITC-labeled streptavidin for detection. The cellnuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole)reagent. Micrographs were then captured using a Zeiss Microscope(Thornwood, N.Y.) coupled with the Spot imaging program (DiagnosticInstruments, Inc).

Hyaluronan expression in the sections was evaluated by the level ofgreen fluorescent intensity in the sections. While significantpericellular hyaluronan staining was present in tumor sections takenfrom animals treated with vehicle, high dose dexamethasone or low dosedexamethasone, pericellular hyaluronan staining was almost completelyabsent from tumor sections taken from animals treated with PEGPH20alone, PEGPH20+low dose dexamethasone, or PEGPH20+high dosedexamethasone. Hyaluronan staining in tissue samples from animalstreated with PEGPH20 alone was virtually the same as the hyaluronanstaining in tissue samples from animals treated with PEGPH20 incombination with either low or high dose dexamethasone indicating thatdexamethasone did not alter the activity of PEGPH20 on the removal ofpericellular hyaluronan.

B. BxPC3 Pancreatic Cancer Xenograft Models

To further examine if dexamethasone affected the antitumor or hyaluronandegrading activities of PEGPH20, human pancreatic cancer xenograftmodels were evaluated.

1. Antitumor Activity in BxPC3 Pancreatic Cancer Xenograft Models

Tumor cells from the BxPC3 pancreatic cancer cell line were grown toapproximately 80% confluency, trypsinized, collected, washed once inHBSS (Hank's balance salt solution, Mediatech Inc.), and re-suspended in50% Matrigel in HBSS at 2×10⁷ cells/mL on ice before inoculation intoanimals. Athymic female nude mice (average weight ˜20 g) were inoculatedintramuscularly (IM) with 0.05 mL of cell suspension, peritibially, inthe left hind leg (adjacent to the tibia periosteum). The tumors in theanimals were assessed twice a week and allowed to grow to a mean tumorvolume of approximately 250 mm³ (Day −1; Table 13). Actual tumor volumeswere determined using VisualSonics Vevo 770 high-resolution ultrasound.

The animals were randomly grouped into 6 groups of 8 animals per group(6+2 satellite mice) and treated with vehicle (API buffer), PEGPH20(157,500 Units/kg, 4.5 mg/kg), low dose dexamethasone (1.25 mg/kg), highdose dexamethasone (5 mg/kg), PEGPH20 (4.5 mg/kg)+low dose dexamethasone(1.25 mg/kg) and PEGPH20 (4.5 mg/kg)+high dose dexamethasone (5 mg/kg).The dose and frequency of the PEGPH20 (or vehicle) intravenousinjections was 157,500 Units/kg or approximately 4.5 mg/kg, on Days 0,3, 7, 10, 13 and 17 for a total of 6 injections starting on Day 0 (dosevolume of 0.1 mL). The doses and frequency of the dexamethasoneintraperitonal injections was 1.25 mg/kg at the low dose and 5 mg/kg atthe high dose, once a day until Day 17 (QDx17) with a dose volume of 0.1mL.

The tumor volumes of all mice were measured pre-treatment on Days −4 and−1 (mean tumor volume of ˜250 mm³) and on Days 4, 7, 11, 14 and 17 bycapturing images using the VisualSonic ultrasound system and using anultrasound imaging software program to calculate the tumor volume (Table13). The percent tumor growth inhibition (% TGI) was calculated asdescribed above in Example 9A.

The data in Table 13 below summarizes the results. The results show thatthe affects on tumor volume with concurrent administration ofdexamethasone, either high or low dose, with PEGPH20 was similar to theresults when PEGPH20 was administered alone. The data from the BxPC3pancreatic cancer xenograft models show that dexamethasone did notinterfere with PEGPH20-mediated tumor growth inhibition.

TABLE 13 Tumor Volume and Statistical Analysis of Antitumor Activity ofPEGPH20 and Dexamethasone as Single-agents and in Combination. Day GroupDay −4 Day −1 Day 4 Day 7 Day 11 Day 14 Day 17 API-buffer 201.7 281.0517.4 598.4 772.2 974.1 1266.2 SE 33.30 36.74 70.35 60.11 81.22 114.56172.61 PEGPH20 4.5 mg/kg 196.87 275.75 350.44 394.40 473.50 596.96790.16 SE 34.06 47.53 65.68 73.09 92.44 112.96 160.10 p-value 0.05 0.030.02 0.02 0.03 % TGI 68 63 60 54 48 Dexamethasone 5 mg/kg 197.29 231.18396.26 379.39 481.54 603.82 720.95 SE 34.61 44.61 82.79 62.01 66.9495.93 115.49 p-value 0.01 0.01 0.01 0.02 % TGI 53 49 46 50 Dexamethasone1.25 mg/kg 202.85 312.65 463.51 609.40 732.92 796.84 933.86 SE 38.2448.31 55.42 104.30 95.85 100.99 120.33 Dexamethasone 5 mg/kg + 205.98317.51 394.98 414.21 516.62 674.16 794.21 PEGPH20 4.5 mg/kg SE 40.6841.62 45.53 63.27 75.87 87.85 114.50 p-value 0.03 0.02 0.03 0.02 % TGI70 59 49 52 Dexamethasone 1.25 mg/kg + 207.68 265.19 325.49 341.39424.14 527.05 610.31 PEGPH20 4.5 mg/kg SE 41.97 56.80 60.12 73.10 63.4175.65 81.90 p-value 0.03 0.01 0.003 0.003 0.003 % TGI 74 76 68 62 65 SE= Standard Error; % TGI = % Tumor Growth Inhibition; TR = TumorRegression; p-value = One-tailed Paired Equal Variance Student's T-test

2. Effect of Dexamethasone on PEGPH20 Plasma Enzyme

Plasma samples were also collected from individual PC3 tumor-bearingsatellite mice of control and treatment group mice 24 hours after thefinal PEGPH20 and dexamethasone dose at Day 17. Plasma PEGPH20Hyaluronidase activity was assessed using the method described inExample 4. Animals treated with PEGPH20, PEGPH20+low dose dexamethasoneand PEGPH20+high dose dexamethasone all had similar levels of plasmahyaluronidase activity indicating that dexamethasone did not interferewith the level of PEGPH20 activity in the plasma of these mice.

Plasma samples were additionally used to measure soluble plasmahyaluronan in the control group and each of the treatment groups (R&DSystems Catalog #DY3614; ELISA assay). In the vehicle control group, theplasma hyaluronan levels ranged from 2,000 to 4,000 ng/mL. The high dosedexamethasone group had a high range of variability in plasma hyaluronanconcentrations (one high concentration and one low concentration butboth measurable) but the low dose dexamethasone group had plasmahyaluronan levels ranging between 2,000 to 4,000 ng/mL, similar to thecontrol animals. Mice treated with PEGPH20 alone or withPEGPH20+dexamethasone (either high or low dose) had no detectable (belowlimit of quantitation) hyaluronan demonstrating that dexamethasone didnot alter the PEGPH20 plasma activity in these mice.

The data from the BxPC3 pancreatic cancer xenograft models show thatdexamethasone also had no effect on PEGPH20 activity in the plasma inthis model.

3. Effect of Dexamethasone on PEGPH20 Hyaluronan Degradation Activity inBxPC3 Pancreatic Cancer Xenograft Models

Tumor tissues from the two satellite mice from all 6 treatment groups inExample 9B.1 above, were harvested by tissue biopsy on Day 18 andstained for the presence of hyaluronan. Tumor biopsies were fixed in 10%NBF for 48 hours, embedded in paraffin, and sectioned. Samples wereprobed for hyaluronan using a biotinylated HA-binding protein (HABP),then probed with FITC-labeled streptavidin for detection. The cellnuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole)reagent. Micrographs were then captured using a Zeiss Microscope(Thornwood, N.Y.) coupled with the Spot imaging program (DiagnosticInstruments, Inc).

Hyaluronan expression in the sections was evaluated by the level ofgreen fluorescent intensity in the sections. While significantpericellular hyaluronan staining was present in tumor sections takenfrom animals treated with vehicle control, high dose dexamethasone, orlow dose dexamethasone, hyaluronan staining was almost completely absentfrom tumor sections taken from animals treated with PEGPH20 alone,PEGPH20+low dose dexamethasone, or PEGPH20+high dose dexamethasone.Hyaluronan staining in tissue samples from animals treated with PEGPH20alone was virtually the same as the staining in tissue samples fromanimals treated with PEGPH20 and either low or high dose dexamethasoneindicating that similar to the results in the PC3 xenograft model,dexamethasone did not alter the activity of PEGPH20 on the removal ofhyaluronan, in the BxPC3 xenograft model.

Example 10 The Effect of Dexamethasone on Musculoskeletal Effects inHumans

This Example describes a study assessing the effect of dexamethasone onameliorating or reducing the adverse musculoskeletal events thatresulted from the administration of PEGPH20 in humans. Dexamethasone wasadded to a dosing regime as a premedication to eliminate or amelioratethe musculoskeletal effects of PEGPH20 administration. The treatmentcycle was defined as a 28-day period, with PEGPH20 administeredintravenously (IV) and dexamethasone administered orally. Dosing ofPEGPH20 and dexamethasone took place on days 1, 4, 8, 11, 15, 18, 22 and25 of the 28-day cycle. On each dosing day, a premedication regimen of 4mg of dexamethasone was administered orally one hour prior to thePEGPH20, followed by a second dose of 4 mg dexamethasone 8-12 hoursafter PEGPH20 dosing.

A. Inclusion and Exclusion Criteria

Patients were enrolled in the study after signing the informed consentform. The patients had at least one confirmed, advanced, solid tumor;were refractory to standard treatment; had at least one tumor that wasmeasurable by RECIST criteria; had a Karnofsky performance status ≧70%;had an ejection fraction of ≧50%, as determined by echocardiogram atbaseline; had a life expectancy of at least 3 months; was not pregnantand agreed to use contraception; and had acceptable organ function asshown by hematology, hepatic, renal and coagulation laboratory assays.

The patients additionally had none of the following exclusion criteria:known brain metastasis; New York Heart Association Class III or IVcardiac disease, myocardial infarction, or cardiac arrhythmia requiringmedical therapy; active infection requiring systemic therapy;uncontrolled diabetes requiring insulin therapy; medical conditionsrequiring heparin therapy; known HIV, hepatitis B or hepatitis Cinfection; known allergy to hyaluronidase; serious nonmalignant disease;or intolerance to dexamethasone. The patients also agreed to comply withthe protocol and not participate in any other concurrent interventionaltherapeutic study. In all cases, the patients entered the program underpain medication. For example, the first patient in this study was takingoxycodon-acetaminophen & oxycontin for pain related to her presentingcondition but not for study drug-related pain.

B. Dosing of and Assessment

1. First Patient

Initially, one patient with advanced solid tumors who satisfied theinclusion/exclusion criteria was dosed with PEGPH20+dexamethasone twiceweekly for 28 days at an initial low dose of 0.5 μg/kg. The firstpatient was a 55-year old, 68.6 kg female patient, with ovarian cancer.

Approximately one hour prior to PEGPH20 administration, dexamethasone (4mg) was administered orally. Then, a needle/catheter of the appropriategauge was placed intravenously and PEGPH20 was administered over 5minutes as a slow IV push. For PEGPH20, dosing was prescribed on a μg/kgbasis, based on the actual patient weight on Day 1. PEGPH20 was suppliedas an aqueous solution containing 3.5 mg/mL PEGPH20 with 10 mM histidineand 130 mM NaCl at a pH of 6.5. The dose was diluted with normal salineto a final volume of 10 mL. The starting dose was 0.5 μg/kg, given twiceweekly for 28 days as specified by the dosing regime. The second dose ofdexamethasone was given 8-12 hours later. NSAIDs (ibuprofen) orcyclobenzaprine were used to treat musculoskeletal pain if it occurred.

During the 28 day cycle, the patient underwent a series of medical andlaboratory assessments. A schedule of these assessments and theirtime-points is shown in Table 14. All data collected at each time-pointwas recorded on a Case Report Form.

TABLE 14 Schedule of Assessments Activity Screening Treatment Cycle 1Study Day (Cycle Day) −10 to −1 1 2/3 4 8 11 15 18 22 25 Signed informedconsent X Inclusion/Exclusion criteria X Medical history X Medicalhistory - 28 days X Physical examination X Karnofsky performance statusX Vital signs (BP, HR, RR, X X^(de) X X X X X X X Temperature) Bodyweight X^(d) Target PE X^(d) X^(d) Tumor biopsy tissue sample for HA XX^(b) staining 12-lead ECG X^(d) Echocardiogram X X^(d) Clinicalchemistry, CBC, and X X^(d) X^(d) X^(d) X^(d) coagulation parametersInflammatory markers - ESR and X^(cd) X CRP Urinalysis X X^(cd) X^(d)Pregnancy test X Tumor markers as appropriate X X^(d) HA catabolitesX^(cd) X X^(cd) X^(cd) X^(cd) X^(cd) X^(cd) X^(cd) X^(cd) PEGPH20Immunogenicity plasma X^(d) X sample PK blood sample collection X^(cd) XX X X X X X X Efficacy imaging/radiologic X evaluation ADC-MRI, DCE-MRI,and/or X X^(a) X^(a) FDG-PET PEGPH20 infusion X^(e) X^(e) X^(e) X^(e)X^(e) X^(e) X^(e) X^(e) Concomitant medications X X X X X X X X Adverseevents X X X X X X X X X ^(a)To be performed on day 3 (optimally) or day4 prior to infusion and at end of cycle 1 (after day 25). ^(b)Post-dosetumor biopsy for HA staining if possible, at any time day 2 or beyond.^(c)After administration ^(d)Before administration ^(e)Duringadministration F/U—Follow-up

Adverse events or side effects, including musculoskeletal events andother adverse events, were assessed. Any undesirable medical event wasconsidered an Adverse Event (AE) if its onset occurred during or afterthe patient's first exposure to PEGPH20 in combination withdexamethasone but no later than 30 days after the last dose (whether ornot it was considered related to PEGPH20+dexamethasone).

AEs were categorized by severity (the measure of intensity as opposed tothe measure of seriousness) by the Investigator using the NCI CTCAEVersion 4.0 and the guidelines presented in Table 15 below. All AEs wereadditionally classified by the Investigator depending on if the AE wereRelated, Probably Related, Possibly Related, Unlikely Related or NotRelated specifically to the administration of PEGPH20 and/ordexamethasone.

TABLE 15 Classification of Adverse Events by Severity SeverityDefinition Mild (Grade 1) Symptoms or signs may exist, but are transientand easily tolerated. Intervention is not indicated. Moderate (Grade 2)Symptoms limit some activities of daily living. Minimal, local, ornoninvasive intervention is indicated to avert patient discomfort.Severe (Grade 3 or Symptoms are incapacitating. Hospitalization ofhigher) other urgent intervention may be indicated.

The patient also was monitored for dose-limiting toxicity (DLT). Toqualify as a DLT, the adverse event had to have been considered relatedto PEGPH20+dexamethasone treatment and must have emerged during thefirst 28 days of therapy. DLT was defined as any National CancerInstitute (NCI) Common Terminology Criteria for Adverse Events (CTCAE)Grade 3 or higher (see Table 15) non-hematologic toxicity. Exceptions tothis included nausea and vomiting that occurred without prophylacticanti-emetic therapy but were effectively treated by such therapy, andGrade 3 musculoskeletal toxicities that were readily treated such thatthey were downgraded to Grade 2 or less, or resolved within 24 hours.DLT was also defined as any ongoing and persistent Grade 2 toxicity thatfailed to resolve over 21 days and that limited the patient's ability tocomply with the protocol therapy. A final DLT definition included anyGrade 4 or prolonged Grade 3 hematological toxicity.

After dosing with PEGPH20 in combination with dexamethasone at day 1 andday 4, the patient experienced Grade 1, bilateral knee pain. The 3^(rd)dose on day 8 was given without complication. The bilateral knee paincontinued to be Grade 1, was intermittent, and did not require therapyor limit ambulation. A physical examination of the knee indicated thatthere were no physical changes or limitations in movement of thearticulation. The adverse event was not considered to be a DLT.

The patient completed the regimen (4 weeks, biweekly dosing on days 1,4, 8, 11, 15, 18, 22 and 25) with no additional related complications.In addition, the patient also had diarrhea, abdominal distention (Grade3; not related) and actinic rash (Grade 2) on bilateral upper and lowerarms (Grade 2; not related) The patient was discontinued and exited thestudy due to clinical disease (ovarian cancer) progression.

2. Second Patient

Since the dosing regime of PEGPH20 in combination with dexamethasone waswell-tolerated in the first patient, the dose of PEGPH20 was escalatedin a second patient with esophageal cancer. The patient, male and 71years of age, was dosed with 1.6 μg/kg PEGPH20+dexamethasone twiceweekly for 28 days. Except for the higher dose, the dosage regimen wasthe same as in the first patient. Medical and laboratory assessments,including adverse events and DLTs were conducted as described above forthe first patient.

Grade 1 musculoskeletal side effects were observed. The adverse effects,all Grade 1, included mild achiness in the lower back, fatigue, achinessand pain in the left hip, muscle cramps in the hands and feet. No DLTswere reported. The patient had adverse events that included fatigue(Grade 1; related), left hip ache (Grade 1; not related to study drug);muscle cramps and lower back achiness (Grade 1; possibly related tostudy drug).

The patient continued to cycle 2, where the patient received weeklydoses. The patient discontinued due to disease progression on Day 15 ofCycle 2.

3. Third Patient

A third patient was dosed at 5.0 μg/kg PEGPH20+dexamethasone twiceweekly for 28 days. The first dose was received without complications.On the day of the second dose, the patient had Grade 1 muscle cramps inthe hands, which progressed to Grade 3 muscle cramping in the hands,legs feet and toes after the third dose. The patient did not receive anyadditional infusions and was discontinued from the study.

4. Fourth and Fifth Patients

Due to the Grade 3 muscle cramping experienced after a third dose of 5.0μg/kg PEGPH20, the dose was reduced to 1.6 μg/kg in two patients, eachreceiving 1.6 μg/kg PEGPH20+dexamethasone twice weekly for 28 days.

The fourth patient, who had moderately differentiated colonadenocarcinoma, experienced Grade 1 bilateral cramping in the fingers,hands, feet and calves. Notably, a post-dose biopsy of a metastaticlesion of the liver demonstrated a reduction in pericellular HA whencompared to an archived tumor biopsy (see Example 11). The patientcompleted Cycle 2, but was withdrawn from the study prior to startingcycle 3 due to disease progression.

The fifth patient, who had moderately differentiated colorectaladenocarcinoma, reported Grade 1 intermittent bilateral cramping in thethighs starting the same day as the second PEGPH20 dose, which wasongoing. Grade 1 intermittent left knee pain also occurred on a singleday, which was one day after the second PEGPH20 dose and re-occurred asGrade 1 on the day the sixth PEGPH20 dose was given. This patient alsoreported Grade 2 intermittent bilateral cramping in the hands andmuscles of the lower extremities, which started the same day as thefourth PEGPH20 dose.

C. Summary of Results

For the study of patients dosed with less than 5.0 μg/kg PEGPH20 incombination with dexamethasone, the adverse effects were mostlymusculoskeletal in nature, including bilateral knee pain, right footmuscle cramping, left and right hand muscle cramping, which are allstudy drug related and are all Grade 1. The five patients werediscontinued due to disease progression. One patient, receiving 1.6μg/kg twice weekly, completed two cycles of therapy and then wasdiscontinued due to disease progression.

In sum, the results show that the use of dexamethasone premedication, 4mg administered pre- and post-PEGPH20 dosing on the dosing day (one hourprior to and eight to 12 hours post-PEGPH20 administration), hasattenuated the severity of musculoskeletal events. Thus, the use ofdexamethasone permits tolerable administration of PEGPH20 at higherdosing and dosing frequency than achieved by PEGPH20 in the absence ofdexamethasone.

Example 11 Histochemical Detection of HA

Samples for histochemical detection of HA were obtained from apre-biopsy tumor specimen and a post-cycle 1 metastatic liver biopsysample from a patient dosed for 4 weeks with 1.6 μg/kgPEGPH20+dexamethasone as described in Example 10. An archived pre-dosebiopsy (pre biopsy) obtained in 2007 (3.5 years prior to the PEGPH20study) and a post-PEGPH20 Cycle 1 (3 days after the last dose) biospsy(Post biopsy) were obtained from a female colon cancer patient withliver metastases. The patient post-treatment biopsy was obtained afterone cycle of PEGPH20 treatment at 1.6 μg/kg on a twice weekly schedulewith dexamethasone co-treatment.

Briefly, the tumor biopsies were fixed in normal buffered formalin (NBF)and 5 μm sections cut and stained using a biotin labeled hyaluronanbinding protein (HABP-bio) (Seikagaku, Japan). After washing to removethe primary reagent, a labeled secondary reagent was used. Nuclei werecounter-stained using a DAPI (4′,6-diamidino-2-phenylindole) reagent.Micrographs were captured via a Nikon Eclipse TE2000U invertedfluorescent microscope coupled to a Insight FireWire digital camera(Diagnostic Instruments, Michigan) or ZEISS overhead scope (Carl Zeiss,Inc.) that has the same imaging system.

The histochemical staining of the samples with biotinylated-HA bindingprotein demonstrated a decrease in pericellular and stromal HA levelsafter one cycle of PEGPH20 treatment. The results are summarized inTable 16. The H score represents the relative intensity of pericellularand stromal HA. The data demonstrates the ability of PEGPH20 to degradetumor-associated HA.

TABLE 16 Histochemical Detection of HA Pericellular tumor Stroma cells(% cells stained) (% area stained) % total area Specimen 3+ 2+ 1+ 0 H 3+2+ 1+ 0 H Tumor Stroma** prebiopsy 10 30 25 35 115 30 50 15 5 205 40 50postbiopsy 0 0 25 75 25 30 30 23 17 173 20 5 **tumor associated stroma

Example 12 HPLC Method for the Estimation of Hyaluronan (HA) Level inPlasma

This Example describes a method for the determination of theHA-disaccharide content in plasma. The method employs the hydrolysis ofHA with Chondroitinase ABC to release the HA-disaccharides, derivatizethem with 2-amino acridone (AMAC) and analyze them on a reverse-phaseHPLC coupled with fluorescence detection. Quantitation of theHA-disaccharides is accomplished by comparison with HA-disaccharidestandards.

1. Working Standards

In the method, a working standard solution was generated. First, adilute stock solution (DSS) was generated from an HA-disaccharide StockSolution (SS). The HA disaccharide SS was generated by adding 1 mL ofwater to a vial of HA-Disac (V-labs, Cat. No. C3209) containing 2 mg oflyophilized powder to make a uniform suspension. To generate dilutestock solutions, 5 W of the SS solution was diluted with 125 μl of waterto generate a DSS1 solution (containing 200 pmoles/μl HA-Disac; 200nmoles/ml HA-Disac). Five-fold serial dilutions in water were made togenerate DSS2 (containing 40 pmoles/μl HA-Disac; 40 nmoles/ml HA-Disac)and then DSS3 (containing 8 pmoles/μl HA-Disac; 8 nmoles/ml HA-Disac).Next, working standard solutions were generated as set forth in 25%human serum albumin (HAS) (ABO Pharmaceuticals, Cat. No. 1500233) orNormal Mouse plasma (Bioreclamation, Cat. No. MSEPLEDTA2-BALB-M) as setforth in Tables 17 and 18.

TABLE 17 Working Standard Solution in HSA HA0Disac (pmoles WSS# DSS3DSS2 DSS1 Water (μl) 25% HSA (μl) in 150 μl) WSS0 0.00 130.00 20.00 0WSS1 1.25 128.70 20.00 10 WSS2 3.13 126.87 20.00 25 WSS3 6.25 123.7520.00 50 WSS4 12.50 117.50 20.00 100 WSS5 6.25 123.75 20.00 250 WSS612.50 117.50 20.00 500 WSS7 6.25 123.75 20.00 1250 WSS8 12.50 117.5020.00 2500 WSS9 25.00 105.00 20.00 5000 WSS10 50.00 80.00 20.00 10000

TABLE 18 Working Standard Solution in Normal Mouse Plasma Normal MouseHA0Disac Plasma (pmoles WSS# DSS3 DSS2 DSS1 Water (μl) (μl) in 150 μl)WSS0 0.00 50.00 100.00 0 WSS1 1.25 48.70 100.00 10 WSS2 3.13 46.87100.00 25 WSS3 6.25 43.75 100.00 50 WSS4 12.50 37.50 100.00 100 WSS56.25 43.75 100.00 250 WSS6 12.50 37.50 100.00 500 WSS7 6.25 43.75 100.001250 WSS8 12.50 37.50 100.00 2500 WSS9 25.00 25.00 100.00 5000 WSS1050.00 00.00 100.00 10000

2. Hydrolysis and Derivation of Samples

Next, the sample was hydrolyzed. The sample (e.g. plasma) was preparedby taking approximately 100 μg of protein in a polypropylene tube andadjusting the volume to 340 μl with water. A matrix blank also wasprepared by taking dilution buffer (1.59 g HEPES, 5.07 g NaCl, 1800 mLwater, pH 7.0) equal to the volume of the sample and adjusting thevolume to 340 μl. Hydrolysis of the samples and matrix blank wereeffected by adding 60 μl of TFA to the sample tube and matrix blank tubeand the contents were mix and incubated at 100° C. for 4 hours. Thevials were allowed to cool to room temperature. The vials wereevaporated to dryness using a speed vac. Then, 300 μl of water was addedto each tube and vortexed to resuspend the samples.

For derivation of hydrolyzed samples, blanks and working samples, 45 μlof each sample (sample, blank or working sample) was evaporated todryness in a speed vac. Then, 10 μl of SAS was added to the driedsample, blank and working standards. Then, 50 μl ABA/NaCNBH3 labelingsolution was added. The tubes were vortexed and centrifuged briefly.Then, 440 μl of Mobile Phase A was added and the tubes were mixed well.Mobile Phase A was prepared as follows: 132 mL of 1 M ammonium acetatebuffer (Sigma, Cat. No. A7330) was added to a 1 L volumetric flask andwater added to fill the flask. Following derivation, nominal on-columnloads per 20 μl of injection for the working standards is as set forthin Table 19.

TABLE 19 Fuc GalN GlcN Gal Man WSS # (pmol) (pmol) (pmol) (pmol) (pmol)WSS1 25 3.0 105 42 175 WSS2 30 3.7 127 51 211 WSS3 35 4.3 150 60 250WSS4 41 5.0 173 69 287 WSS5 46 5.6 195 78 325

3. HPLC

The HPLC column was equilibrated at a flow rate of 1.0 mL/min with theinitial mobile phase settings as outlined in Table 20. The system wasallowed to equilibrate until the baseline was steady. HPLC analysis wasperformed with the instrument parameters as outlined in Table 20.

TABLE 20 HPLC Instrument Parameters Parameter Values Column BakerbondC18 reversed phase column, 4.6 × 250 mm, 5 um Column Temperature RoomTemperature Mobile Phase A 0.2% n-butylamine, 0.5% phosphoric acid, 1%tetrahydroruran in water Mobile Phase B 50% mobile phase A, 50%acetonitrile Flow Rate 1.0 mL/min Injection volume 20 μl DetectorFluorescence; Excitation 360 nm, Emission 425 nm Sample condition 4-6°C. Gradient Time (min) % A % B 0.0 95 5 25.0 95 5 50.0 85 15 50.1 0 10060.0 0 100 60.1 95 5 70.0 95 5

The sequence for sample analysis was the following: WSS5 (1 injection)for column conditioning/equilibration/detector gain; water injection (1injection); WSS3 (3 injections); WSS1 (1 injection); WSS2 (1 injection);WSS4 (1 injection); WSS5 (1 injection); Water (1 injection); MatrixBlank (1 injection); Sample 1 (1 injection); Sample 2 (1 injection);WSS3 (3 injections); Water (1 injection). The system was consideredsuitable when there was acceptable separation quality; the signal tonoise ratio for the shorter monosaccharide peak in the WSS1 sample wasequal to or more than 10; the relative standard deviation (RSD) of thepeak areas for each monosaccharide standard for the 6 injections of WSS3was equal or less than 4%; the correlation coefficient (r) was 0.99 (rwas measured using software to plot the peak area of each workingstandard against the on-column load (expressed as pmol) using the firstthree injections of the WSS3 standard and calculating the slope,intercept and correlation coefficient for the working standards using alinear least square regression model); the peak areas for peakscorresponding to monosaccharides were no more than 2% of the peak areameasured for WSS5; and the peak areas for peaks corresponding tomonosaccharides in water injection were no more than 0.5% of the peakareas measured for WSS5.

4. Sample Analysis

The average corrected peak area for each monosaccharide in each samplepreparation was determined. Valley-to-valley integration was used forthe GalN peak. To determine this, the linear curves generated from theworking standards were used to calculate the amount of eachmonosaccharide loaded for each sample preparation. For each type ofmonosaccharide, the average molar ratio of monosaccharides per proteinmolecule for each sample was calculated. Then, for each sample, theoverall sum of the average molar ratios for all five monosaccharides wasdetermined. The calculations were performed based on the following:Molecular weight (MW) of non-glycosylated hyaluronidase protein is 51106g/mol; the total volume of each sample was 500 ml; the sample dilutionfactor is 0.15; the volume of each injection is 20 μl; and theconversion factor from mg to pg is 10⁹. The calculations were performedas follows for each monosaccharide:

The amount of monosaccharide for each preparation was calculated usingthe following equation:

${{Monosaccharide}\left( {p\;{mol}} \right)} = \frac{{{Peak}\mspace{14mu}{Area}} - {Intercept}}{Slope}$

The number of monosaccharides per protein molecule was calculated bysing the following formula:

${{Monosaccharides}\mspace{14mu}{per}\mspace{14mu}{protein}\mspace{14mu}{ratio}} = \frac{{Monosaccharide}\mspace{14mu}\left( {p\;{mol}} \right) \times \mspace{70mu}{MW} \times 500\mspace{14mu}{µl}}{0.1\mspace{14mu}{mg} \times 10^{9} \times 20\mspace{14mu}{µl} \times 0.15}$

For each sample, the results for each sample were reported as themonosaccharides per protein ratio for each monosaccharide along with thesum of the five monosaccharide ratios.

Example 13 Pharmacokinetics of PEGPH20 with or without Dexamethasone

In the studies in humans described in Example 6 (PEGPH20) and Example 10(PEGPH20+dexamethasone), blood samples were collected at scheduled timesfrom the patients. Plasma samples were stored frozen, and concentrationsof PEGPH20 were determined by measuring hyaluronidase activity using amodified turbidimetric assay as described in Example 4. The USPturbidimetirc method used for the determination of hyaluronidaseactivity awas based on the formation of an insoluble precipitate thatoccurs when HA binds with acidified serum. PEGPH20 concentrations wereexpressed as units of hyaluronidase activity (U/mL) as interpolated froma calibration curve. Activity was reported to the nearest 1 U/mL, andthe analytical methods were applicable for measuring plasmahyaluronidase concentrations as low as 0.3125 U/mL.

1. PEGPH20

Blood samples from patients in Example 6 that received one to three IVdoses of PEGPH20 at doses that ranged from 0.5 μg/kg to 50 μg/kg wereanalyzed for pharmacokinetics of PH20.

For the two patients that initially received a single dose of 50 μg/kg(Example 6B.1), the plasma concentration over time was similar. MaximalPEGPH20 concentrations in the plasma were measured soon after infusion,and steadily disappeared from circulation over time. Pharmacokineticanalysis indicated a small distribution volume, slow plasma clearanceand terminal half-life of approximately 2 days. The results show thatinitially following administration, approximately 30 U/mL of enzyme wasmeasured in the plasma. The level of plasma enzyme steadily decreasedover time, but was detectable 24 and 48 hours after administration withless than approximately 2 U/mL present at 72 hours after administration.The PK parameters revealed a plasma half life from 28-48 hours in thepatients. Table 21 sets forth a summary of the PK parameters includingmaximum observed plasma concentration (Cmax; U/mL), absolute/systemicclearance (CL; mL/hr/kg), distribution half-life (t_(1/2)α), eliminationhalf-life (t_(1/2)β), and volume of the central compartment (V₁; mL/kg).

TABLE 21 PK Parameters After Single 50 μg/kg Dose of PEGPH20 Cmax CLt_(1/2)α t_(1/2)β V₁ Patient (U/mL) (mL/hr/kg) (hr) (hr) (mL/kg) 1 30.52.44 4.77 49.4 57.96 2 31.3 3.29 2.47 25.9 55.93

For the remaining twelve patients in the study treated with single ormultiple doses of PEGPH20 that ranged from 0.5 μg/kg to 1.5 μg/kg,plasma concentrations were measurable after dosing (but less than orabout 1.5 U/mL), but concentrations fell below the limit ofquantification (BQL) during the first day. Because plasma concentrationswere typically near or below the limit of quantification, sufficientinformation on the time course of plasma concentrations was notavailable. Generally, there was a dose-dependent increase in systemicexposure for the first day after IV dosing with PEGPH20 at low dosesranging from 0.5 μg/kg to 1.5 μg/kg.

For the eight patients that received multiple doses in the study inExample 6, measurable plasma concentrations were detected after repeatadministration in four of these eight patients. These plasmaconcentrations were low ranging from 0.34 to 0.83 U/mL and were similarto those detected after the initial dosing.

2. PEGPH20+Dexamethasone

Blood samples from patients in Example 10 that received multipleadministrations of PEGPH20 at doses that ranged from 0.5 mg/kg to 5.0mg/kg were analyzed for pharmacokinetics of PH20.

To facilitate comparison to the pharmacokinetics of PEGPH20 withoutdexamethasone described above, plasma concentration vs. time data wascollected prior to repeat administration of PEGPH20. For patientstreated with 0.5 μg/kg or 1.6 μg/kg, maximum plasma concentrations wereconsistent with those observed in the study without dexamethasone.Concentrations fell to the limit of quantification within the first day.The maximum concentration measured from the single patient treated with5.0 μg/kg PEGPH20 was approximately 1/10 of the Cmax values detected forthe patients treated with 50 μg/kg PEGPH20 in the study withoutdexamethasone. These results show that plasma pharmacokinetics ofPEGPH20 is similar in the presence or absence of dexamethasone.

Example 14 Pharmacodynamics of PEGPH20 with or without Dexamethasone

The enzymatic activity of PEGPH20 was measured by monitoringconcentrations of hyaluronan present in the circulation. A disaccharideassay described in Example 12 was used to measure the concentrations ofHA and its catabolites in serial plasma samples that were collected atscheduled times from patients in the studies in humans described inExample 6 (PEGPH20) and Example 10 (PEGPH20+dexamethasone).

1. PEGPH20

Blood samples from patients in Example 6 that received one to three IVdoses of PEGPH20 at doses that ranged from 0.5 μg/kg to 50 μg/kg wereanalyzed for HA catabolites. Plasma HA concentrations prior to PEGPH20dosing were typically less than 1 μg/mL for all patients in the study.

For the patients that received a single 50 μg/kg dose of PEGPH20, plasmaconcentrations of hyaluronan increased significantly. Despite therelatively more rapid disappearance of PEGPH20 from the plasma (Example12.1), elevated concentrations of HA appeared to accumulate more slowlyand persist for a period greater than 2 weeks (over 400 hours). Theprolonged effect on HA catabolism, i.e. sustained HA concentrations inthe circulation, is consistent with the enzymatic activity of PEGPH20 ina peripheral compartment or extravascular tissue.

For the remaining 12 patients that were treated with single or multipledoses of PEGPH20 that ranged from 0.5 μg/kg to 1.5 μg/kg, HA catabolitelevels increased in a dose-dependent manner over the course of a week.Maximal HA concentrations (C_(max)) and one-weekarea-under-the-curve-estimates (AUC_(0-168h)) were also determined foreach patient to quantify the pharmacodynamic response. The resultsshowed that systemic exposure to HA catabolites, as measured by maximumplasma concentration or area-under-the-curve, appeared to increase withincreasing dose of PEGPH20.

2. PEGPH20+Dexamethasone

Blood samples from patients in Example 10 that received multipleadministrations of PEGPH20 at doses that ranged from 0.5 μg/kg to 5.0μg/kg were analyzed for HA concentrations. Consistent with theobservations in the samples from patients receiving only PH20 describedabove, plasma HA concentration vs. time data increased afteradministration of PEGPH20. Concentrations of plasma HA measured duringthe first week of dosing increased with increasing dose of PEGPH20. Inthe three patients that completed a full cycle of treatment and received8 doses of PEGPH20, the results showed sustained increased plasma HAconcentrations in samples from all three patients measured throughoutthe dosing period.

Example 15 Magnetic Resonance Imaging

Diffusion weighted MRI was performed using a single shot spin-echosequence to estimate pixel-by-pixel values for the apparent diffusioncoefficient. Dynamic contrast enhanced magnetic resonance imaging(DCE-MRI) included imaging during infusion with a contrast agent.Calibration was accomplished using a two part phantom containing aninner tube and ice/water mixture. Scans were performed pre-treatment andpost-treatment.

1. Apparent Diffusion Coefficient Magnetic Resonance Imaging (ADC-MRI)

Apparent diffusion coefficient magnetic Resonance imaging (ADC-MRI)measures the volume of water that has moved across the cell membranebased upon a calculation derived from the pre- and post-treatment scans.ADC-MRI scans were completed for a total of 10 of the 14 patients in thestudy described in Example 6 and 4 of the 5 patients in the studydescribed in Example 10. Analysis of the images acquired from eachpatient was performed by a radiologist at Imaging Endpoints (Scottsdale,Ariz.), and quantitative estimates of ADC were computed for tissues ineach patient. A summary of the ADC-MRI findings associated with tumorregions is shown in Table 22. As shown in the Table, increases inADC-MRI were observed in 7 of 14 (50%) of patients following PEGPH20dosing. Increased ADC values are consistent with the mechanism of actionof PEGPH20. ADC values, however, did not change in 5 of 14 patients, andvalues decreased in 2 of 14 patients.

TABLE 22 ADC-MRI Summary Post-Dose Change in Tumor ADC- Dose & FrequencyScan Days MRI from baseline Example 6 50 μg/kg D4 no change 0.5 μg/kg;2x/wk D3 no change 0.5 μg/kg; 21 day cycle D3 increase 0.5 μg/kg; 21 daycycle D4 decrease in lymph nodes 0.5 μg/kg; 21 day cycle D3 increase0.75 μg/kg; 21 day cycle D3 increase 0.75 μg/kg; 21 day cycle D3 nochange 0.75 μg/kg; 21 day cycle D3, D30 increase 1.0 μg/kg; 21 day cycleD5 increase 1.5 μg/kg; 21 day cycle D3 no change Example 10 1.6 μg/kg +D3, D29 increase dexamethasone; 2x/wk 5.0 μg/kg + D1, D4 increase D1dexamethasone; 2x/wk 1.6 μg/kg + D2, D25 decrease D25 dexamethasone;2x/wk 1.6 μg/kg + D1, D2 no change dexamethasone; 2x/wk

2. Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI)

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) measuresblood flow that indicates a change in tumor's vascularity. Scans werecompleted in 4 of 5 patients in the study described in Example 10.Analysis of images acquired from each patient was performed by aradiologist at Imaging Endpoints (Scottsdale, Ariz.), and quantitativeestimates of the volume transfer coefficient (Ktrans), blood volume (Vp)and extracellular volume fraction (Ve) were computed for tissues in eachpatient. A summary of the DCE-MRI findings associated with tumor regionsis set forth in Table 23. Significant increases in the Ktrans parameterwere observed in the two patients that were scanned on the day ofPEGPH20 dosing. The increase in Ktrans within hours of dosing isconsistent with preclinical data that show PEGPH20 causes vasculardecompression and increased blood flow (Thompson et al. (2010) Mol.Cancer Ther., 9:3052-64.

TABLE 23 DCE-MRI Summary Post-Dose Change in Tumor DCE- Dose & FrequencyScan Days MRI from baseline 1.6 μg/kg + D3, D29 decrease in ktrans atD29 dexamethasone; 2×/wk 5.0 μg/kg + D1, D4 increase in ktrans, Ve, Vpdexamethasone; 2×/wk (8 hr). Return to baseline (D4) 1.6 μg/kg + D2, D25No baseline scan available. dexamethasone; 2×/wk Increase in Vp on D25vs. D2. No change in Ktrans 1.6 μg/kg + D1, D2 Increase in Ktrans (8 hr,dexamethasone; 2×/wk 24 hr. Increase in Ve for lung tumor but not livertumor (D1, D2). No change in Vp

Example 16 Interspecies Scaling Algorithm

In preclinical models, it was observed that PEGPH20, either alone or incombination with gemcitabine effectively inhibited tumor growth at dosesas low as 0.01 to 0.1 mg/kg. To determine the equivalent human exposureat these doses, an interspecies scaling algorithm was used that assumesplasma clearance of PEGPH20 scales in proportion to body weight. Basedon this, it was found that the efficacious doses in mice of 0.01 to 0.1mg/kg scale to human equivalent doses of 0.75 μg/kg to 7.5 μg/kg. Asmice have approximately 20 times the circulating levels of HA comparedto humans, an equivalent dose of PEGPH20 can have relatively moreanti-tumor activity in patients.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

The invention claimed is:
 1. A method for ameliorating in a subject anadverse musculoskeletal effect from an administered hyaluronidase,comprising: a) systemically administering a hyaluronidase to thesubject, wherein: the hyaluronidase is a PEGylated soluble human PH20hyaluronidase; the subject is human; and the adverse musculoskeletaleffect is selected from among: an adverse effect that is Grade 3 orGrade 4; and an adverse effect that is ongoing or persistent Grade 2toxicity that fails to resolve over a course of treatment with thehyaluronidase and that limits the ability of the subject to comply withthe hyaluronidase therapy; the grade of the adverse musculoskeletaleffect is defined by Common Terminology Criteria for Adverse Events(CTCAE) scale; and b) administering a sufficient amount of aglucocorticoid to the subject to ameliorate the adverse musculoskeletaleffect.
 2. A method for ameliorating in a subject an adversemusculoskeletal effect from an administered hyaluronidase, comprising:a) prior to administering the hyaluronidase, administering a sufficientamount of a glucocorticoid to the subject to ameliorate an adversemusculoskeletal effect of the hyaluronidase when it is administered,wherein: the subject is human; the adverse musculoskeletal effect isselected from among: an adverse effect that is Grade 3 or Grade 4; andan adverse effect that is ongoing or persistent Grade 2 toxicity thatfails to resolve over a course of treatment with the hyaluronidase andthat limits the ability of the subject to comply with the hyaluronidasetherapy; and the grade of the adverse musculoskeletal effect is definedby Common Terminology Criteria for Adverse Events (CTCAE) scale; andthen b) systemically administering the hyaluronidase, wherein thehyaluronidase is a PEGylated soluble human PH20 hyaluronidase.
 3. Themethod of claim 1, wherein the glucocorticoid is selected from among acortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisolone and a prednisone.
 4. The method of claim 1, wherein theglucocorticoid is a dexamethasone.
 5. The method of claim 1, wherein theglucocorticoid is administered orally.
 6. The method of claim 1, whereinthe adverse musculoskeletal effect is Grade 3 or Grade 4 and is selectedfrom among one or more of muscle and joint pain and stiffness, stiffnessof upper extremities, stiffness of lower extremities, cramping, musclesoreness and tenderness over the entire body, weakness, fatigue and adecrease in range of motion at knee and elbow joints.
 7. The method ofclaim 2, further comprising administering a sufficient amount of aglucocorticoid concurrent with or subsequent to administration of thehyaluronidase.
 8. The method of claim 1, wherein the glucocorticoid isco-administered with the hyaluronidase.
 9. The method of claim 1,wherein the glucocorticoid is administered prior to administration ofthe hyaluronidase.
 10. The method of claim 1, wherein administration ofthe glucocorticoid is at least 1 or more hours prior to administrationof the hyaluronidase.
 11. The method of claim 1, wherein theglucocorticoid is administered subsequent to administration of thehyaluronidase.
 12. The method of claim 1, wherein administration of theglucocorticoid is at least 8 hours to 12 hours after administration ofthe hyaluronidase.
 13. The method of claim 1, wherein the glucocorticoidis administered prior to and after administration of the hyaluronidase.14. The method of claim 1, wherein the amount of glucocorticoidadministered is between 0.1 to 20 mgs, inclusive.
 15. The method ofclaim 1, wherein the amount of glucocorticoid administered is between0.4 to 20 mgs, inclusive.
 16. A method of ameliorating in a subject anadverse musculoskeletal effect from an administered hyaluronidase,comprising: a) intravenously administering the hyaluronidase, wherein:the hyaluronidase is a PEGylated soluble human PH20 hyaluronidase; thesubject is human; and the adverse musculoskeletal effect is selectedfrom among: an adverse effect that is Grade 3 or Grade 4; and an adverseeffect that is ongoing or persistent Grade 2 toxicity that fails toresolve over a course of treatment with the hyaluronidase and thatlimits the ability of the subject to comply with the hyaluronidasetherapy; the grade of the adverse musculoskeletal effect is defined byCommon Terminology Criteria for Adverse Events (CTCAE) scale; and b)administering a sufficient amount of a glucocorticoid to the subject toameliorate the adverse musculoskeletal effect.
 17. The method of claim1, wherein the hyaluronidase is administered several times a week, twicea week, once a week, every 21 days or once monthly.
 18. The method ofclaim 1, wherein the hyaluronidase is administered to treat a hyaluronanassociated disease or condition.
 19. The method of claim 18, wherein thehyaluronan associated disease or condition is selected from among onethat is associated with high interstitial fluid pressure, a cancer,edema, disc pressure and an inflammatory disease.
 20. The method ofclaim 19, wherein the disease or condition is edema and the edema iscaused by organ transplant, stroke or brain trauma.
 21. The method ofclaim 19, wherein the disease or condition is an inflammatory diseaseand the inflammatory disease is selected from among Rheumatoidarthritis, scleroderma, periodontitis, psoriasis, atherosclerosis,chronic wounds, Crohn's disease, ulcerative colitis and inflammatorybowel disease.
 22. A method of ameliorating in a subject an adversemusculoskeletal effect in a subject from an administered hyaluronidase,comprising: a) systemically administering a hyaluronidase to the subjectin an amount to treat a cancer that is a tumor, wherein: thehyaluronidase is a PEGylated soluble human PH20 hyaluronidase; thesubject is human; and the adverse musculoskeletal effect is selectedfrom among: an adverse effect that is Grade 3 or Grade 4; and an adverseeffect that is ongoing or persistent Grade 2 toxicity that fails toresolve over a course of treatment with the hyaluronidase and thatlimits the ability of the subject to comply with the hyaluronidasetherapy; the grade of the adverse musculoskeletal effect is defined byCommon Terminology Criteria for Adverse Events (CTCAE) scale; and b)administering a sufficient amount of a glucocorticoid to the subject toameliorate the adverse effect.
 23. The method of claim 19, wherein thedisease or condition is a cancer and the cancer is selected from amongany one or more of a late-stage cancer, a metastatic cancer and anundifferentiated cancer.
 24. The method of claim 22, wherein the tumoris selected from among ovarian cancer, in situ carcinoma (ISC), squamouscell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small celllung cancer, breast cancer, brain cancer and colon cancer.
 25. Themethod of claim 22, wherein administration of the hyaluronidase iseffected intravenously (IV), subcutaneously, intramuscularly,intradermally, transdermally or sub-epidermally.
 26. The method of claim1, wherein: the subject has cancer; and the adverse musculoskeletaleffect results from systemic administration of the human hyaluronidase.27. The method of claim 1, wherein: the adverse musculoskeletal effectresults from intravenous administration of a single dose of a PEGylatedhyaluronidase that is 0.00005 mg/kg to 5 mg/kg of the mass of thesubject to whom it is administered; and the amount of hyaluronidase is0.0005 mg/kg to 5 mg/kg of the mass of the subject to whom it isadministered.
 28. The method of claim 1, wherein: the adverse effectresults from administration of an amount of PEGylated hyaluronidase in arange between 0.3 Units/kg to 320,000 Units/kg, inclusive, of the massof the subject; and the amount of the hyaluronidase administered is in arange between 0.3 Units/kg to 320,000 Units/kg, inclusive, of the massof the subject.
 29. The method of claim 1, wherein: the adverse effectresults from administration of an amount of PEGylated hyaluronidase thatis between 0.3 Units/kg and 320,000 Units/kg, inclusive, of the mass ofthe subject; the amount of a hyaluronidase administered is between 0.3Units/kg and 320,000 Units/kg, inclusive, of the mass of the subject;and the amount of glucocorticoid administered is between 0.4 to 20 mgs,inclusive.
 30. The method of claim 1, wherein the human PH20hyaluronidase is a truncated form thereof lacking a C-terminalglycosylphospatidylinositol (GPI) attachment site or a portion of theGPI attachment site.
 31. The method of claim 30, wherein the PH20 ortruncated form thereof is administered in a composition comprising apolypeptide having the sequence of amino acids set forth in any of SEQID NOS:4-9.
 32. The method of claim 1, wherein the glucocorticoid isdexamethasone.
 33. The method of claim 22, further comprisingadministration of a cancer treatment.
 34. The method of claim 33,wherein the cancer treatment is selected from among surgery, radiation,a chemotherapeutic agent, a biological agent, a polypeptide, anantibody, a peptide, a small molecule, a gene therapy vector, a virusand DNA.
 35. The method of claim 1, wherein: the hyaluronidase isneutral active and soluble when secreted from a cell; and thehyaluronidase polypeptide is a C-terminal truncated PH20 selected from:(a) a polypeptide that consists of the sequence of amino acids 36-467,36-468, 36-469, 36-470, 36-471, 36-472, 36-473, 36-474, 36-475, 36-476,36-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483, 36-484, 36-485,36-486, 36-487, 36-488, 36-489, 36-490, 36-491, 36-492, 36-493, 36-494,36-495, 36-496, 36-497, 36-498, 36-499 or 36-500 of SEQ ID NO:1; or (b)a polypeptide consisting of a sequence of amino acids that exhibits atleast 85% sequence identity to a polypeptide of a).
 36. The method ofclaim 1, wherein: the hyaluronidase is neutral active and soluble whensecreted from a cell; and the hyaluronidase polypeptide is a C-terminaltruncated PH20 selected from: (a) a polypeptide that consists of thesequence of amino acids set forth in any of SEQ ID NOS: 4-9; or (b) apolypeptide that consists of a sequence of amino acids that exhibits atleast 95% sequence identity to any of SEQ ID NOS: 4-9.
 37. The method ofclaim 36, wherein the PH20 polypeptide consists of a sequence of aminoacid residues having at least 98% sequence identity to residues 36-483of SEQ ID NO:1.
 38. The method of claim 1, wherein: the adversemusculoskeletal effect results from administering the hyaluronidase inan amount that is between 0.1 μg/kg to 1 mg/kg, inclusive, of the massof the subject to whom it is administered; and the amount ofhyaluronidase administered is between 0.1 mg/kg to 1 mg/kg, inclusive,of the mass of the subject to whom it is administered.
 39. The method ofclaim 1, wherein: the adverse musculoskeletal effect results fromadministering the hyaluronidase in an amount that is between 16 to16,000 Units/kg, inclusive, of the mass of the subject to whom it isadministered; and the amount of hyaluronidase administered is between 16to 16,000 Units/kg, inclusive, of the mass of the subject to whom it isadministered.
 40. The method of claim 2, wherein: the adversemusculoskeletal effect results from administering the hyaluronidase inan amount that is between 0.1 μg/kg to 1 mg/kg, inclusive, of the massof the subject to whom it is administered; and the amount ofhyaluronidase administered is between 0.1 μg/kg to 1 mg/kg, inclusive,of the mass of the subject to whom it is administered.
 41. The method ofclaim 2, wherein the hyaluronidase polypeptide consists of the sequenceof amino acids set forth in any of SEQ ID NOS: 4-9, or consists of asequence of amino acids that exhibits at least 95% sequence identity toany of SEQ ID NOS: 4-9.
 42. The method of claim 16, wherein: the adversemusculoskeletal effect results from administering the hyaluronidase inan amount that is between 0.1 μg/kg to 1 mg/kg, inclusive, of the massof the subject to whom it is administered; and the amount ofhyaluronidase administered is between 0.1 μg/kg to 1 mg/kg, inclusive,of the mass of the subject to whom it is administered.
 43. The method ofclaim 16, wherein the hyaluronidase polypeptide consists of the sequenceof amino acids set forth in any of SEQ ID NOS: 4-9, or consists of asequence of amino acids that exhibits at least 95% sequence identity toany of SEQ ID NOS: 4-9.
 44. The method of claim 22, wherein: the adversemusculoskeletal effect results from administering the hyaluronidase inan amount that is between 0.1 μg/kg to 1 mg/kg, inclusive, of the massof the subject to whom it is administered; and the amount ofhyaluronidase administered is between 0.1 μg/kg to 1 mg/kg, inclusive,of the mass of the subject to whom it is administered.
 45. The method ofclaim 22, wherein the hyaluronidase polypeptide consists of the sequenceof amino acids set forth in any of SEQ ID NOS: 4-9, or consists of asequence of amino acids that exhibits at least 95% sequence identity toany of SEQ ID NOS: 4-9.
 46. The method of claim 2, wherein theglucocorticoid is administered orally.
 47. The method of claim 16,wherein the glucocorticoid is administered orally.
 48. The method ofclaim 22, wherein the glucocorticoid is administered orally.
 49. Themethod of claim 1, wherein the subject is identified as having anadverse musculoskeletal effect or having had an adverse musculoskeletaleffect from a previous administration of the hyaluronidase.
 50. Themethod of claim 1, wherein the adverse musculoskeletal effect ismusculoskeletal pain.
 51. The method of claim 1, wherein the adversemusculoskeletal effect is difficulty standing or difficulty walking orboth.
 52. The method of claim 1, wherein the glucocorticoid isadministered to the human subject before the hyaluronidase.
 53. Themethod of claim 52, wherein the glucocorticoid is dexamethasone.
 54. Themethod of claim 1, wherein the adverse musculoskeletal effect is Grade4.
 55. The method of claim 1, wherein the adverse musculoskeletal effectis ongoing or persistent Grade 2 toxicity that fails to resolve over acourse of treatment with the hyaluronidase and that limits the abilityof the subject to comply with the hyaluronidase therapy.
 56. The methodof claim 1, wherein the adverse musculoskeletal effect is Grade
 3. 57.The method of claim 2, wherein the adverse musculoskeletal effect isGrade
 4. 58. The method of claim 2, wherein the adverse musculoskeletaleffect is ongoing or persistent Grade 2 toxicity that fails to resolveover a course of treatment with the hyaluronidase and that limits theability of the subject to comply with the hyaluronidase therapy.
 59. Themethod of claim 2, wherein the adverse musculoskeletal effect is Grade3.
 60. The method of claim 16, wherein the adverse musculoskeletaleffect is Grade
 4. 61. The method of claim 16, wherein the adversemusculoskeletal effect is ongoing or persistent Grade 2 toxicity thatfails to resolve over a course of treatment with the hyaluronidase andthat limits the ability of the subject to comply with the hyaluronidasetherapy.
 62. The method of claim 16, wherein the adverse musculoskeletaleffect is Grade
 3. 63. The method of claim 22, wherein the adversemusculoskeletal effect is Grade
 3. 64. The method of claim 22, whereinthe adverse musculoskeletal effect is Grade
 4. 65. The method of claim22, wherein the adverse musculoskeletal effect is ongoing or persistentGrade 2 toxicity that fails to resolve over a course of treatment withthe hyaluronidase and that limits the ability of the subject to complywith the hyaluronidase therapy.
 66. The method of claim 1, wherein thehyaluronidase is administered intravenously.
 67. The method of claim 2,wherein the hyaluronidase is administered intravenously.
 68. The methodof claim 22, wherein the hyaluronidase is administered intravenously.